Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis.We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. We have conducted a comprehensive analysis of the murine cytomegalovirus and host cell transcriptomes during lytic infection. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. Infection alters expression of many unexpected host genes, including many noncoding RNA genes. Correspondingly, many cluster in unexpected biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. Betherpesvirinae subfamily, are a group of species-specific herpes viruses that establish life-long infection of their hosts. Human cytomegalovirus (HCMV) can cause devastating disease and death in congenitally-infected infants, and long-term neurological complications in survivors. In adults, HCMV can cause a spectrum of diseases in immune compromised patients involving multiple organs and tissues and is a primary cause of graft loss in transplant patients The cytomegaloviruses, classified within the There exist a number of challenges to our understanding of CMV pathogenesis as well as progress in vaccine and antiviral drug development. Two outstanding challenges are the gaps in our knowledge of viral genes and how these gene products interact with host cellular gene products to cause disease. Despite the publication of the first sequence of the HCMV genome in 1990 MCMV is the most widely used model to study HCMV diseases and recapitulates many of clinical and pathological findings found in human diseases. Our understanding of MCMV viral genes and genomes has evolved with the technology used to study them. A major milestone in understanding MCMV came with decoding the first MCMV complete genome sequence by Rawlinson and colleagues in vitro and in vivo passage. Also, Lacaze and colleagues Subsequent refinements in the annotation of the MCMV were introduced by classical molecular and biochemical studies that are reflected in the current NCBI reference sequence. The application of new technologies to study the MCMV genome emerged in the last decade and include proteomic et al.Altogether, these new technologies have refined and advanced our knowledge of viral genes and the MCMV genome. Nevertheless, we still lack definitive annotation for the standard lab strains of MCMV and specific knowledge of how many of these genes function during natural infection and disease. Currently, two annotations of MCMV genomes are used – the original Rawlinson's annotation with minor modifications (GenBank accession no. GU305914.1) where 170 open reading frames (ORFs) are identified and the NCBI reference sequence annotation (GenBank accession no: NC_004065.1) with 160 ORFs. We previously used a transcriptomic approach to analyze gene products of HCMV We describe MCMV transcriptional products that differ from predicted ORFs, novel spliced transcripts, and novel transcripts derived from intergenic regions of the genome. Additionally, we found that the most abundant viral transcript (MAT) is a spliced transcript recently identified as a noncoding RNA that limits accumulation of cellular miRNAs In this study, we set out to complete a transcriptomic analysis of MCMV infection. We analyzed viral transcripts through classical cDNA cloning and sequencing and through next generation sequencing of cDNA generated from total cellular RNA (RNA-Seq). Analysis of cDNA libraries is a well-proven approach to analyze viral transcripts based on isolation of long transcripts, molecular cloning of the transcripts, and traditional Sanger-based sequencing of the cDNA clones. Traditional cloning has many advantages, including isolation of novel transcripts that may not be identified by probe-based technologies, as well as precise analysis of transcript splice sites and transcript 3′ ends. The introduction of massively parallel sequencing techniques represents a major new technology to study gene expression. Basically, RNA  is converted to a library of smaller cDNA fragments. Adaptors are added to the fragments, and the shorter fragments are sequenced in a high-throughput manner using next generation sequencing technology. This RNA-sequencing (RNA-Seq) approach is free of selection biases associated with traditional cloning or probe-based methods and allows for the entire transcriptome to be analyzed in a quantitative manner  at 9 time points after infection. For RNA-Seq analysis, RNAs collected at the same 9 time points were pooled, converted to cDNA, and sequenced on the Illumina Genome Analyzer IIx. Of the 33,995,400 reads that passed the filter from infected cells, 11% aligned to MCMV genome indicating a 585-fold coverage of the viral genome.Dataset S1.A total of 448 cDNA clones were included in the final analyses . Generally, temporal assignment of cDNA clones in this study agrees with previous studies and a detailed comparison, including discrepancies to earlier studies is provided in andTable S1, and a complete list of the 448 cDNA clones isolated in this study and their characteristics are presented in Table S2. A detailed comparison of transcripts cloned in this study compared to current annotations (GU305914.1 and NC_004065) is presented in Table S3, and includes estimated relative expression measured as reads per kilobase per million mapped reads (RPKM) derived from RNA-Seq analysis.As shown in Analysis of the cDNA clones revealed many novel transcripts with the following characteristics:Novel antisense transcripts. Excluding 20 cDNA clones that mapped to intergenic regions, 275 (64%) of cDNA clones were in the sense (S) orientation, 39 (9%) were antisense (AS), and 114 (27%) overlapped more than one gene in both S and AS orientations relative to original annotation provided by Rawlinson and colleagues Transcripts overlapping more than one currently annotated genes. Several cDNA clones were isolated that overlap more than one currently annotated gene. For example, four cDNA clones in our library overlapped both the m15 and m16 genes. The longest of these clones specifies a 1673 bp transcript, whereas current annotation predicts two genes of 908 bp (m15) and 632 bp (m16). These data suggest the possibility that several ORFs are translated from unexpectedly long or polycistronic transcripts. Unusually long and polycistronic transcripts have recently been shown to be a feature of HCMV transcriptome The absence of transcripts in currently annotated regions. Several gene regions were not well represented in the cDNA cloning study. For example, no cDNA clones overlapping m01 or m170 were found in this study, and these regions had the lowest RPKM reads by RNA-Seq of 132 and 141, respectively (gray histograms in Table S3), consistent with earlier gene array-based studies m04 and m138, both represented with multiple clones in our cDNA library, have RPKM values of 14,137 and 16,935 respectively. Because some viral transcripts with higher RPKM reads were not represented in the classical cDNA library , we conclude the cDNA library cloning did not capture all viral transcripts. Nevertheless, the failure to identify clones in regions poorly represented by RNA-Seq data suggests that further attention is required to prove or disprove the existence of genes predicted by in silico (ORF) analysis.Novel spliced transcripts. One of the most striking aspects of the cloning study was the abundance of novel spliced transcripts. A total of 22 novel spliced transcripts were cloned in this study as well as spliced transcripts reported by others. A complete list of spliced transcripts identified in this and other studies is provided in Table S4. One of the most abundant novel spliced transcripts identified in this study maps to the M116 gene (8 cDNA clones). Current annotation for M116 predicts a protein of 645 AA, whereas the clones in this study predict a novel truncated protein product. Splicing results in a change of open reading frame at amino acid residue 350 of the currently annotated M116 protein and introduction of a new stop codon at position 401 (Dataset S2). By far the most abundant novel spliced transcript is that overlapping m169 in the S orientation, and m168 in the AS orientation and is discussed in detail below.m169. This antibody was used in immunoblot analysis of cell lysates prepared from mock-infected cells and cells infected with wild-type (WT) BAC-derived Smith strain virus, or various multi-gene and single-gene mutant virus strains. This antibody reacted with a protein of approximately the expected size (17 kDa) , and additional genes of unknown functions (M73 and m15). Second, as shown by comparing m74-M75 , M85-M87 and M88-m90 . Minor differences in abundance of some transcripts can be attributed to differences in the time points analyzed in these two studies as well as the fact that our analysis achieved an order of magnitude greater sequencing depth  using our cDNA clones to generate strand specific riboprobes  and RNA-Seq data alignment to MCMV genome indicates a sudden drop in reads around this nucleotide position. Assuming that transcripts in this region are co-terminal, band sizes predict transcript initiation sites in m11, m12, m13 or m14, and m15 . We therefore propose that m19 should be removed from MCMV genome annotation. This is consistent with our cloning study where 4 transcripts have been isolated in this region and none overlap m19 in the sense orientation . Evidence from the cDNA library and RNA-Seq alignment . The band of approximately 2 kb  transcript with an 81 bp intron. Northern blot analysis . Additionally, northern analysis detected another less abundant band of approximately 3 kb. Leatham and colleagues UL119-115 genes. While we failed to isolate cDNA clones overlapping m117 region, we predict the larger, less abundant 3 kb band observed initiates in m117, though additional northern or 5′RACE studies are needed to confirm the start site of the larger transcript.Next we analyzed gene regions in which we detected novel spliced transcripts. The m72-m74 region was previously shown to have a very complex transcriptional profile m60, m73 and M73.5 spliced transcripts. In our cloning study four isolated clones correspond to M73.5 transcripts  and one to M73 (L33) . Transcripts corresponding to m60 were not isolated in the cloning study, however the RNA-Seq profile in the region corresponding to m60 exon1 shows active transcription . We have also detected a 1.1 kb band , however more analyses are needed to determine its exact 5′ start site. Northern analysis revealed a band of 0.5 kb  could correspond to the unspliced version of E180. All probes used in this region detected bands transcribed from negative genomic strand that correspond to those previously reported by Rapp et al.  as well as previous reports m75 and ends in m72 and corresponds to transcript encoding gH while the 3 kb transcript starts in m74 and ends in m72 and corresponds to transcript encoding dUTPase . Additional very large transcripts transcribed from plus or minus genomic strands detected by the L42 and L147 probes, respectively, have yet to be characterized but underscore the complex transcriptional patterns in this region.The M100-M103 region confirmed previously published findings. We have detected single M102 transcript from plus genomic strand as described by Scalzo Figure S5A). A probe derived from M100 detected a single transcript from negative genomic strand corresponding to M100 Figure S5B). The presence of sense and antisense transcripts in this gene region corresponds to findings for HCMV M103 gene region we detect 2 transcripts from plus genomic strand that correspond to those described by Lyons et al Figure S5C). Temporal expression of transcripts detected by northern in this region is in line with our cDNA analysis and previously published data Last, we set out to confirm novel antisense transcripts detected in the cDNA library. Analysis of transcription in Based on northern analyses of 5 regions, we conclude that our cDNA and RNA-Seq analyses faithfully represents the MCMV transcriptome in infected primary fibroblasts and confirms the presence of novel transcripts. Moreover, the distribution of clones in the IE, E and L cDNA libraries accurately reflected the accumulation of transcripts detected by northern analyses.Table S5). The top induced, upregulated, repressed and downregulated genes are presented in –Interferon β (Ifnb1) and the interferon-inducible pyhin1  were among the top induced genes, consistent with the expected host response to virus infection. Also congruous with expected host responses to infection were two highly induced genes associated with apoptosis induction (Hrk and Tnfsf10 [a.k.a. TRAIL]). Interestingly, transcription factors  were among the most strongly induced by MCMV infection.RNA-Seq analysis also enabled us to investigate changes in the host transcriptome. Differentially expressed (DE) murine genes in MCMV-infected cells compared to mock-infected cells were identified by calculating RPKM. This analysis identified 10,748 statistically significant (p<0.05) genes altered by infection . There were also a surprising number of genes involved in development, differentiation, and stem cell renewal strongly induced or upregulated by infection, including FoxA1, Spint1, Lin 28B, En2, Gabrq, Esx1, Trim71, Trp73, Cpne5, Cdh7, Cited 1, Pou4f1, and Jag2. The relevance of these genes, as well as others including Art3, Ugt8, and Trank1 to infection is not clear. We analyzed protein levels of several induced and upregulated transcripts whose relevance to MCMV infection is unknown . MCMV infection also caused repression or downregulation of noncoding (nc)RNAs including the small nucleolar RNA gene, Snord15A and genes of unknown function including 3 long intergenic noncoding RNAs (lincRNAs), the miscellanous RNA, 4930412B13Rik, and 2 antisense transcripts . To summarize, while many of the top upregulated genes are associated with host responses to infection, the function of many of the top downregulated and repressed genes during infection are obscure.Interestingly, the top repressed and downregulated genes are primarily of unknown relevance to infection, though many are receptor or cell surface molecules (Table S6A). These were also top networks when the subset of up- and down-regulated genes were evaluated (Table S6B). Also identified were networks associated with cell morphology and hematological system development and function. When this analysis was conducted with only induced and repressed genes, the top networks included cellular development, cell-mediated immune response, cellular function and maintenance, gene expression and embryonic development (Table S6C). The relationships among the molecules in top networks for differentially regulated and induced/repressed genes are shown in Figures Figure S6 and Figure S7. Thus, an unexpected outcome of this analysis is that MCMV infection influences a subset of networks controlling development..Genes and their products do not work in isolation but rather form pathways and networks. Even small perturbations in gene expression in a pathway can exert profound influences on eventual processes or functions. Therefore, we analyzed gene lists for shared common pathways. As expected, the top scoring gene networks for all differentially expressed (DE) genes included (i) infectious disease, antimicrobial response, inflammatory response (28 focus molecules); (ii) inflammatory response, cellular development, cell-mediated immune response (27 focus molecules) and (iii) cell morphology, hematological system development and function, inflammatory response (19 focus molecules)  enrichment using GOrilla ranked lists analysis We report a comprehensive analysis of the MCMV transcriptome during lytic infection derived from cloning and sequencing of viral transcripts and next generation sequencing (RNA-Seq). By combining the approaches of RNA-Seq and traditional cDNA cloning as well as northern and RT-PCR analyses in certain complex regions, we were able to construct a comprehensive profile of viral and host transcription during lytic infection. We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, pathway analysis, and gene ontology analysis.The major findings are as follows: 1) The MCMV transcriptome diverges substantially from that predicted by current annotation; 2) the identification of a novel viral protein specified by the MAT transcript indicates that this transcript functions as an mRNA and a non-coding RNA; 3) the majority of the most abundantly transcribed viral genes are of unknown function; and 4) the host response to infection includes regulation of many host genes and gene networks of unknown relevance to infection.There are four major findings from the analysis of the MCMV transcriptome. First, we demonstrate novel transcripts of MCMV including novel splice variants, transcripts that map to noncoding regions, and transcripts overlapping multiple genes. Earlier, we reported similar novel transcripts of HCMV through analysis of a classical cDNA library Herpesviridae family differ in the extent of antisense transcription during lytic infection.In line with previous studies Second, we observed similar inconsistencies between transcriptomic data and gene annotation for MCMV as previously reported for HCMV Third, analysis of the MCMV transcriptome revealed the striking abundance of the spliced MAT transcript. This gene is also largely conserved in wild isolates of MCMV , also known to be induced by HCMV We also report that lytic infection elicits a profound cellular response in fibroblasts. This study identified 10,748 differentially regulated genes. As the number of mouse genes is estimated to be 33,207 m131/m129 genes Inflammatory chemokine ligand genes are also highly upregulated during infection. MCMV encodes virally-derived chemokine homologs specified by the Insm1). Recently, Insm1 was found to be strongly upregulated by HSV-1 infection and shown to promote HSV gene expression, probably by binding the HSV infected cell protein (ICP)0 promoter. Gabrq. Glutamate receptor signaling was also identified as significantly impacted canonical pathways in our dataset. In the developing brain GABA and glutamate receptors influence neuronal proliferation, migration, differentiation or survival processes Numerous transcription factors are also induced or upregulated by infection including insulinoma-associated 1  and influenza virus, and have been suggested to impact host defenses and innate immunity Trp53 mutation develop rhabdomyosarcomas at high frequency This analysis also revealed immunological disease, cardiovascular disease, genetic disorders and skeletal and muscular disorders as top bio-functions connected with genes altered by MCMV infection. While MCMV involvement in cardiovascular disease is a subject of intensive research, potential involvement in skeletal and muscular disorders is not well documented but may be relevant to the novel observation that MCMV infection of mice with a heterozygous A primary caveat of RNA-Seq analysis is determining whether changes in gene transcript levels are also reflected at the protein level. This is particularly important as herpesviruses can control protein accumulation at the post-transcriptional, translational, and post-translational levels To summarize, this study has refined the understanding of MCMV gene expression and identified new areas of research to advance our understanding of the host response to these ancient viruses. We describe what is to our knowledge, the first herpes virus transcript that functions as both a noncoding RNA that limits accumulation of cellular miRNAs, and an mRNA that specifies a protein. This study also revealed novel features of the host response to infection. Perhaps most importantly, this study identified many virus and host genes of unknown function that are regulated during infection. It is highly likely that further study of these genes may lead to breakthroughs in the understanding and treatment of cytomegalovirus-related diseases.Primary mouse embryonic fibroblasts (MEFs) from BALB/c or Balb.K mice were prepared and maintained as described Smith MCMV infected cBalb/c MEFs were harvested 72 h post infection and viral DNA was isolated as described 5′-GCGGCCGCTTAATTAACC(T)15-3′). After second-strand synthesis, an EcoRI-PmeI adapter was added to the 5′ end and cDNAs were cleaved with EcoRI and PacI. The EcoRI-PmeI adapter was generated by annealing following oligonucleotides: 5′-AATTCCCGCGGGTTTAAACG-3′ and 5′-Pho-CGTTTAAACCCGCGGG-3′. cDNA fragments were inserted into a modified pcDNA3.1(+) previously digested with EcoRI and PacI and transformed into XL1-Blue Supercompetent E. coli cells .RNA was extracted from Smith MCMV infected Balb/c MEF at 4, 8 and 12 hrs after infection (IE library); 16, 24 and 32 hrs after infection (E library); and 40, 60 and 80 hrs after infection (L library). No drug was used to select for different temporal classes of transcripts and equal amounts of RNA from each time point were pooled prior to library construction. cDNA libraries were generated as described previously for HCMV Mse I-digested genomic MCMV DNA was labeled using a DIG High Prime DNA Labeling Detection Starter Kit II (Roche Applied Science) and used to identify virally-derived cDNA clones. Plasmids harboring cDNA clones that reacted with probe were isolated and sequenced from the 5′ end using T7 primer for pcDNA3.1(+) or the 3′ ends using primer (5′GCACCTTCCAGGGTCAAGGAAG) or standard poly (T) primers at the OSU Plant-Microbe Genomics Facility. Sequences were compared to the MCMV Smith strain genome [GenBank accession no. NC_004065] using mega BLAST.Positive selection of viral cDNA clones was performed as described previously 2 petri dishes and exposed to 0.3 PFU/cell of the MW 97.01 strain of murine cytomegalovirus or mock-infected. At 4, 8, 12, 16, 24, 32, 40, 60 and 80 hours after infection, RNA was isolated using TRIZOL Reagent. RNA integrity was assessed on Agilent Bioanalyzer and only samples with RNA index values of at least 9 were used. Equal amounts of RNA from each time point were pooled (0.3 µg of RNA per time point) and treated with DNaseI. Libraries were prepared with Illumina TruSeq RNA kit according to manufacturer's instructions and sequenced on Illumina Genome Analyzer IIx as single-end 36 bp reads. The Illumina TruSeq RNA kit employed does not allow for strand-specific information to be derived from the sequence data. Datasets are available at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) accession no. SRR953479 (sequence reads from MCMV-infected MEFs) and accession no. SRR953859 (sequence reads from mock-infected MEFs).Total RNA was extracted from Balb/c MEF cells cultured in 100 mmhttp://www.broadinstitute.org/igv/) http://sammate.sourceforge.net/) www.ingenuity.com) was used for gene interaction network and canonical pathway analysis. Gene lists were filtered for statistically significant differentially expressed genes (p<0.05) and a fold change cutoff of 2 was set to identify molecules whose expression was significantly differentially regulated. For network generation, these molecules (Network Eligible molecules), were overlaid onto a global molecular network developed from information in the Ingenuity Knowledge Base based on their connectivity. The Functional Analysis of a network identified the biological functions and/or diseases that were most significant to the molecules in the network. Right-tailed Fisher's exact test was used to confirm that biological functions and/or disease assigned to data sets were not due to chance. The nature of individual DE genes was also investigated using the Mouse Genome Informatics databases (http://www.informatics.jax.org/) http://www.ncbi.nlm.nih.gov/gene) Reads were aligned to mouse (NCBI37/mm9 assembly) and MCMV genome (GenBank acc.no. NC_004065.1) using ELAND aligner or Bowtie aligner (for comparison with data provided by Lars Dölken). It is important to note that both ELAND and Bowtie aligners do not map splice junctions and thus give concordant results. Alignments were visualized using Integrative Genomics Viewer  or 10, 30 and 60 hrs after infection. RNA (1 µg/lane or 10 µg/lane (MAT)) was separated by formaldehyde agarose gel electrophoresis and transferred to positively charged nylon membrane and crosslinked by UV irradiation. Membranes were reacted to DIG-labeled probes overnight at 67°C. For MAT detection, a DIG labeled double-stranded DNA probe was made using fragments corresponding to the MAT gene sequences derived from cDNA library clones E1, E125 and E134 using Roche's DIG-High Prime DNA Labeling and Detection Starter Kit I. For all other northern blots, single-stranded DIG-labeled RNA probes were used generated using Roche's DIG Northern Starter Kit. Antisense probes were generated by in vitro transcription from T7 promoter present in pcDNA3.1 plasmids containing cDNA clones that harbor the desired gene fragments . Therefo5′-TTTTTGGATCCATGAGCAACGCGGTCCCGTTC-3′ and R: 5′- TTTTTCTGCAGTCATCACGGGGGGCACCTACC-3′, reacted with BamHI and HindIII (New England Biolabs), inserted into pQE30 expression vector and introduced to E.coli Bl21 pREP4 strain (Qiagen). The protein was induced according to manufacturers' instructions and purified on a His-tag column. Purified protein was used for immunization of Balb/c mice and antibody titer in blood serum was measured by ELISA. When antibody titer in serum reached adequate levels, animals were sacrificed, their spleens isolated and fused with SP2/O cells. Supernatants from motherwells were tested by immunoblot blot on purified MAT protein and positive wells were rescreened by immunoblot using lysates from MEFs infected with WT, Δ7S3, Δ168-169, Δ169-170, Δm168, Δm169 and Δm170 mutants as described below.The m169 gene sequence was amplified by PCR using viral DNA isolated from MCMV BAC pSM3fr using following primers: F: Taq RT-PCR Kit, New England BioLabs). No reverse transcriptase (-RT) controls were run in parallel. Splicing was then verified by PCR amplification using primers that flank putative introns  using Phusion high fidelity polymerase (New England BioLabs).RNA from Mock- or MCMV-infected cells isolated for northern blots was also reverse transcribed using oligo-dT primers (ProtoScript M-MuLV http://rsbweb.nih.gov/ij/).Mock-infected or MCMV-infected primary MEFs, or murine cell lines  were lysed in RIPA buffer. Protein lysates were separated by SDS-PAGE and transferred to PVDF. MAT protein was detected with anti-m169 antibody described above, Jag2 with antibody N-19 (Santa Cruz), Engrailed 2 with En2 PA5-14363 antibody (Thermo Scientific), Trim71 with PA5-19282 (Thermo Scientific), and actin with antibody C4 (Millipore) followed by peroxidase-labeled secondary antibodies (Jackson ImmunoResearch or Abcam). Proteins were visualized using Amersham ECL Prime Western blotting reagent  and quantified using ImageJ software  and Comparison of RPKM values in Marcinowski et al. (2012) and this RNASeq experiment (S1C).(PDF)Click here for additional data file.Dataset S2Spliced cDNA clone overlapping M116 and comparison of predicted protein to current annotation.(PDF)Click here for additional data file.Figure S1RNA-Seq profiles comparison. RNA-Seq data from total RNA obtained from MCMV infected NIH-3T3 fibroblasts from 25 and 48 hrs PI sequenced by Dölken group (GSE35833) was aligned against MCMV genome (gB acc no NC_004065.1) using Bowtie aligner and visualized in IGV in comparison to our RNA-Seq data. The view of the complete genome is shown at the top with 4 areas magnified below (labeled A–D) and the number of reads displayed are noted on the side. Since viral genes display a wide range of expression levels, the whole genome view is shown in wide data range (upper panel) more suitable for displaying highly transcribed regions and a narrowed data range (lower panel) that is more suitable for less transcribed regions. As can be seen, the profiles of the compared alignments are remarkably similar, the only differences being abundance of certain transcripts which are due to different time points analyzed in comparison to the pooled data of our RNA-Seq and significantly greater depth of at least one order of magnitude of our data in comparison to Marcinowski data.(TIF)Click here for additional data file.Figure S2Analysis of the m20–19 region. Balb/c MEF cells were infected with BAC derived Smith virus and harvested 10, 30 and 60 hrs post infection. Total RNA was separated by denaturing gel electrophoresis, transferred to nylon membrane and incubated with probe generated by in vitro transcription from T7 promoter of L57  or probe generated by in vitro transcription from T3 promoter of IE205 transcript [probe should detect m20(S)-m19(AS) transcripts]. RNA integrity and loading was evaluated by inspecting 28S (not shown) and 18S rRNA bands under UV light after transfer to membrane. Predicted genes (Rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cDNA library as well as clones used to generate probes (marked with *). 3′ ends of transcripts are marked with arrowheads. The nucleotide coordinates relative to Smith sequence (NC_004065.1) of isolated transcripts are given below thin arrows, while the names of the clones are written above. Gray histograms showRNA-Seqreads aligned to MCMV genome. Maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots.(TIF)Click here for additional data file.Figure S3Verification of M116 splicing by PCR. Schematic of the M116 gene region and clone L29 (A) and PCR analysis of the splice site (B). In (A) Predicted genes (Rawlinson's annotation) are depicted as empty arrows, while the thin black arrow depicts clone L29 with red arrows depicting the primers used in (B). The 3′ ends of transcripts are marked with arrowheads. The nucleotide coordinates relative to Smith sequence (NC_004065.1). Gray histograms showRNA-Seq reads aligned to MCMV genome. RNA isolated from WT infected Balb/c MEF used in northern blots was reverse transcribed with oligo-dT primers, and then amplified with primers specific for M116 that flank the putative intron (marked by red arrows). No RT controls were run in parallel. Spliced cDNA clones and viral DNA were used as spliced and unspliced amplification controls, respectively.(TIF)Click here for additional data file.Figure S4Analysis of the m71-m75 region spliced transcripts by northern blot and PCR. Balb/c MEF cells were infected with BAC derived Smith virus and harvested at indicated times post infection (A and B). Total RNA was separated on denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for S and AS transcripts. RNA integrity and loading was evaluated by inspecting 28S (not shown) and 18S rRNA bands under UV light after transfer to membrane. Predicted genes (Rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cDNA library as well as clones used to generate probes (marked with *). Images of northern blots are shown using probes derived from the M72 region in (A) and the m74 in (B). The 3′ ends of transcripts are marked with arrowheads. The nucleotide coordinates relative to Smith sequence (NC_004065.1) of isolated transcripts are given below thin arrows, while the names of the clones are written above. Thin gray lines show isolated transcripts that cannot be detected with the probe. Gray histograms showRNA-Seqreads aligned to MCMV genome. Maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots (A and B). In (C), the m71–75 spliced transcript and one of two possible m72–75 spliced transcripts were verified by PCR amplification using primers that flank their putative introns. RNA isolated from WT infected Balb/c MEF used in northern blots was reverse transcribed with oligo-dT primers, and then amplified with primers specific to m71–m75 or m72–m75 spliced transcripts (marked by red arrows). No RT controls were run in parallel. Spliced cDNA clones and viral DNA were used as spliced and unspliced amplification controls, respectively. In (D), the position of exon 1 of m60 Reported by Scalzo et. al. (TIF)Click here for additional data file.Figure S5Northern blot analysis of the M100-M103 region. Balb/c MEF cells were infected with BAC derived Smith virus and harvested at indicated times post infection. Total RNA was separated on denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for S and AS transcripts overlapping M102 (A), M100 (B) and M103 (C) genes. RNA integrity and loading was evaluated by inspecting 28S (not shown) and 18S rRNA bands under UV light after transfer to membrane. Predicted genes (Rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cDNA library as well as clones used to generate probes (marked with *). 3′ ends of transcripts are marked with arrowheads. The nucleotide coordinates relative to Smith sequence (NC_004065.1) of isolated transcripts are given below thin arrows, while the names of the clones are written above. Thin gray lines show isolated transcripts that cannot be detected with the probe. Gray histograms showRNA-Seqreads aligned to MCMV genome. Maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots.(TIF)Click here for additional data file.Figure S6Graphical representation of top 3 genetic networks for differentially regulated genes. Upregulated genes are shown in red, while downregulated are shown in green. Level of differential expression is represented by color saturation with most dramatically changed genes being shown in the most saturated color (strong red or green). These overlapping genetic networks are associated with (i) cell-mediated immune response, cellular development, cellular function and maintenance (30 focus molecules); (ii) infectious disease, antimicrobial response, inflammatory response (27 focus molecules) and (iii) antimicrobial response, inflammatory response, gene expression (24 focus molecules) Click here for additional data file.Figure S7Graphical representation of top 5 genetic networks for genes induced or repressed by infection. Induced genes are shown in red, while repressed are shown in green. Level of differential expression is represented by color saturation with most dramatically changed genes being shown in the most saturated color (strong red or green). These overlapping genetic networks are associated with various developmental processes Click here for additional data file.Table S1MCMV transcripts identified in this study compared to current NCBI Reference Sequence Gene Annotation.(PDF)Click here for additional data file.Table S2cDNA clones isolated in this study and their characteristics.(XLS)Click here for additional data file.Table S3Comparison of experimental and in silico data used for viral gene identification and RPKM values of currently annotated genes.(XLS)Click here for additional data file.Table S4Spliced transcripts of MCMV.(PDF)Click here for additional data file.Table S5Differentially expressed mouse genes with p<0.05 determined by SAMMate with EdgeR.(XLS)Click here for additional data file.Table S6Gene networks and associated genes identified by IPA.(XLS)Click here for additional data file.Table S7GOrilla ranked list analysis of DE mouse genes.(XLS)Click here for additional data file.


Accordingly, a high turnover of the bacterial community was also observed between any two sampling stations (average replacement of 79% of OTU3%), yet no direct correlation with spatial distance was observed within the region. Bacterial community composition and structure differed significantly with increasing water depth along the bathymetric transect. The relative sequence abundance of Verrucomicrobia and Planctomycetes decreased significantly with water depth, and that of Deferribacteres increased. Energy availability, estimated from phytodetrital pigment concentrations in the sediments, partly explained the variation in community structure. Overall, this study indicates a high proportion of unique bacterial types on relatively small spatial scales (tens of kilometers), and supports the sampling design of the LTER site HAUSGARTEN to study bacterial community shifts in this rapidly changing area of the world’s oceans.Knowledge on spatial scales of the distribution of deep-sea life is still sparse, but highly relevant to the understanding of dispersal, habitat ranges and ecological processes. We examined regional spatial distribution patterns of the benthic bacterial community and covarying environmental parameters such as water depth, biomass and energy availability at the Arctic Long-Term Ecological Research (LTER) site HAUSGARTEN (Eastern Fram Strait). Samples from 13 stations were retrieved from a bathymetric  and a latitudinal transect . 454 massively parallel tag sequencing (MPTS) and automated ribosomal intergenic spacer analysis (ARISA) were combined to describe both abundant and rare types shaping the bacterial community. This spatial sampling scheme allowed detection of up to 99% of the estimated richness on phylum and class levels. At the resolution of operational taxonomic units (97% sequence identity; OTU Biogeographic patterns have been identified at global and regional scales for marine microbes, e.g., . In mostA strong impact of spatial distance together with water depth and surface water productivity on variation in marine benthic bacterial community structure has already been detected on a global scale in coastal and deep-sea sediments In this study of the Arctic Long-Term Ecological Research (LTER) site HAUSGARTEN in Fram Strait in situ experiments of bacterial colonization of artificial and deep-sea sediments Fram Strait is the only deep-water connection to the Arctic Ocean. Here, warm Atlantic water masses enter the Arctic Ocean through the West Spitsbergen current, while cold Polar waters exit through the East Greenland Current During the cruise ARK-XXIV/2 in July 2009 with the German research ice-breaker RV Polarstern to the LTER site HAUSGARTEN a and its degradation products phaeopigments, here summarized as chloroplastic pigment equivalents (CPE) Sample processing for all environmental parameters was done as described in 8 bacterial cells as determined by acridine orange direct counting −1  were purified via isopropanol precipitation. Final DNA concentrations ranged from 4–12 ng µl−1.Sediment from the uppermost centimeter originating from three different TV-MUC cores was pooled. Total DNA was extracted from 1 g of this homogenized slurry  and 20–25 ng DNA  in a total volume of 50 µl. Primers were used and PCR amplification (in triplicates per sample), separation of fragments by capillary electrophoresis, evaluation of signals and binning into operational taxonomic units (OTU) was done as described previously ARISA PCR consisted of 1×Eppendorf PCR buffer , 0.25 mM desoxynucleoside-triphosphate mix (Promega), 0.3 g lhttp://vamps.mbl.edu, using primers targeting the V4–V6 region of the bacterial 16 S rRNA gene. SFF files were deposited in the GenBank Sequence Read Archives (www.ncbi.nlm.nih.gov) under BioProject ID: PRJNA208712. Preparation of flowgrams and transformation into an OTU- by- Sample table were conducted with “mothur” http://www.mothur.org in March 2012) and chimeric sequences were identified using the mothur implemented uchime program. Cleaned sequences were clustered at a 97% identity level into operational taxonomic units (OTU3%) and the dataset was normalized by the total amount of sequences per sample to get relative abundances. To investigate the rare biosphere 3% that occurred with only one sequence in the whole denoised dataset (absolute singletons), called SSOabs and b) OTU3% that consisted of only one sequence in at least one sample, and were not absolute singletons (relative singletons or SSOrel), so the total number of sequences for any SSOrel was larger than one 3%, but only 4% of all OTU3% were assigned up to the species level. Therefore we only considered annotation up to genus level for subsequent analyses.Extracted DNA was amplified at the Marine Biological Laboratory  according to the protocol published on 3% . Turnover of OTU was calculated as percentage of pairwise shared, lost or gained OTU relative to the total number of OTU in the two samples. Shared OTU are those appearing in both samples, lost OTU are only present in the first sample and gained OTU are only present in the second sample. To compare bacterial classes found in this study to those found in other studies  veganpermuteMASSExcept for longitude, latitude, spatial distance and water depth, all parameters were normalized by logysis see . DistancBiogeographic patterns of surface sediment bacterial communities were investigated at the Arctic LTER site HAUSGARTEN  with the most abundant classes being Gammaproteobacteria (23%), Deltaproteobacteria (15%) and Alphaproteobacteria (7%). The second most OTU3% abundant phylum was Bacteroidetes (9%) with, among others, the classes Flavobacteria (3%) and Sphingobacteria (5%). Other abundant phyla were Actinobacteria (3%), Acidobacteria (5%), and Verrucomicrobia (4%). Those proportions barely changed when excluding SSOabs from the dataset. These phyla and classes were also found as abundant members of Arctic sediments from the Pacific sector Using MPTS data, a total of 41 phyla, 78 classes, 136 orders, 215 families, and 410 genera were identified . Most of2 = 0.78, p<0.001; determined by linear regression; 2 = 0.85, p<0.001; t distribution with 30 degrees of freedom) and 1.1±0.19 (24 degrees of freedom), were obtained for the comparison with the global dataset and the Siberian margin dataset, respectively. This shows that the best model (i.e. a slope coefficient of 1 and higher explained variance) is obtained in the latter case when only considering sediments from the Arctic.The mean proportions of bacterial classes inhabiting HAUSGARTEN sediments were in very good agreement , water depth  than at the Siberian continental margin (<8 µg cm−3) Chao1 richness estimates were on average 3,010±642 OTU station , which ition −1; . We founer depth  or with 3% with every sampled station was linear . In contrast, the OTU accumulation curve for ARISA data did reach a plateau and only nine stations were needed to recover 95% of all observed OTUARISA (ARISA (here 450) and 16–23 S length identity between different genera or species The increase of newly detected OTUs linear . By sampstations . The OTUOTUARISA . This re3% from phylum to genus, according to 3% could not be assigned to a known phylum. Taking only seven stations into account, at least 95% of all observed phyla, classes or orders were recovered; in contrast, sampling of ten stations was needed to recover 95% of all occurring genera in the dataset  were shared between any two samples at HAUSGARTEN  around the whole globe 3%) with spatial distance between any two samples was observed (p = 0.557), neither for the whole data set, nor for samples of the latitudinal transect  or of the bathymetric transect alone  when based on MPTS data including singletons. Removing absolute singletons from the dataset led to the same conclusions (data not shown). In contrast, community composition of samples from the bathymetric transect based on ARISA – known to detect the more abundant types - significantly correlated with spatial distance .On average 21±2% OTUUSGARTEN  which isabs; 263–2,251 m water depth differences). Pairwise shared OTU3% gradually decreased from 25% to 19% (34%–27% when removing SSOabs) from samples from the shallowest HAUSGARTEN station HG-I to station HG-V  with a gradual increase in dissimilarities of community structure with increasing water depth differences , as assessed by ANOVA of the distances to group centroids abs). These findings indicate that samples taken within a water depth zone were more similar to each other than across the zones. Grouping of the communities indicated higher similarities within the depth zones ∼1000–2000 m and>∼2500 m, which was previously also found for meiofauna taxa densities In a non-metric multidimensional scaling plot (NMDS), visualizing dissimilarities of bacterial communities between samples, those from the bathymetric transect were located further apart from one another and had significantly higher community dispersion as those from the latitudinal transect . The lat3% including singletons  and longitude ; Particle flux of organic matter to the deep sea generally decreases with increasing water depth e.g. . We obseAlthough we did not find a significant correlation between water depth and CPE concentrations  of variation in community structure could be significantly explained , and on average 38±8% per sample. Interestingly, it showed similar responses to water depth changes and CPE concentrations as the whole community: water depth differences were highly correlated with differences in community structure and explained 6% of the variation in the SSOrel community data, CPE concentrations correlated significantly with differences in community structure and explained 2% of the variation in the community . Along the 120-km long latitudinal transect, no increasing bacterial community dissimilarity with increasing spatial distance could be observed. Nevertheless, a turnover of on average 79% OTU3% (still 68% when absolute singletons were removed) was detected between any two samples taken within a distance of on average 13 km. Pigment concentrations as a proxy for energy supply in the form of phytodetritus sedimentation influenced bacterial community richness and structure, but no strong energy-diversity relationship was found within the investigated range. We identified indicator taxa that showed significant changes in relative sequence abundance with changes in water depth or pigment concentrations. This study demonstrates the complexity of bacterial community structure in deep-sea sediments and the necessity to investigate the regional biodiversity of deep-sea life not only at one single spot, but over scales of 1–100 km and different water depth zones, in order to better evaluate community responses related to environmental variations.We found a spatially highly diverse bacterial community in surface sediments of the Long-Term Ecological Research site HAUSGARTEN (Eastern Fram Strait). With 13 sampling stations over an area of about 3,385 kmFigure S1OTU accumulation curves.(DOC)Click here for additional data file.Table S1List of samples taken during the Polarstern cruise ARK-XXIV/2 in 2009 and measured environmental parameters.(DOC)Click here for additional data file.Table S2Comparison of dataset structure based on ARISA and MPTS using Spearman correlation and Procrustes tests.(DOC)Click here for additional data file.Table S3Observed and estimated richness of OTU or taxa at different taxonomic levels and shared OTU or taxa between all stations.(DOC)Click here for additional data file.Table S4Observed and estimated richness of ARISA and MPTS data per station and in the total dataset.(DOC)Click here for additional data file.Table S5Spearman’s correlation matrix of alpha diversity measures, water depth and pigment concentrations (CPE).(DOC)Click here for additional data file.Table S63%(A), OTU3% without SSOabs (B) and OTUARISA (C).Percentages of pairwise shared, lost and gained OTU(DOC)Click here for additional data file.Table S7Community response to water depth at different taxonomic levels.(DOC)Click here for additional data file.Table S83% dataset.Linear and quadratic regression of phyla and classes in the OTU(DOC)Click here for additional data file.Text S1Comparison of ARISA and MPTS and Richness of OTU.(DOC)Click here for additional data file.


Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry ( Osteochondral defects (OCDs) result from traumatic injuries or natural degradation of cartilaginous tissue with aging and encompass serious damage to articular cartilage and/or underlying calcified subchondral bone Tissue engineering provides a promising approach for the treatment of osteochondral defects employing biomaterials, progenitor cells and growth factors in vitro and in vivoin vivo still remain elusive.Growth factors, especially those belonging to the super family of transforming growth factor beta (TGF-β) play multifunctional roles in the context of tissue engineering. TGF-β is involved in chondroinduction both in vivo. Osteochondral tissue regeneration is more complex than individual osseous or chondral tissue regeneration as the scaffold involved needs to support cartilage repair and the chondral interface needs to be supported by underlying subchondral bone in situin vivo are sparse in vivo osteochondral repair is novel. The aim of the present study was to evaluate the osteo- and/or chondro-inductive ability of silk (mulberry and non-mulberry origin) fibroin biomaterials using human bone marrow stromal cells (hBMSCs) in vitro. Silk fibroin scaffolds from mulberry and non-mulberry origin differ in their inherent natural properties and thus comparison of two kinds of silk scaffolds is important. The investigation is further expanded to growth factor guided (TGF-β3 or BMP-2) in vivo cellular infiltration and neo-matrix formation on multi-layered cell-free (acellular) silk scaffolds  in order to demonstrate the feasibility of using two different silk scaffolds as three dimensional (3D) implantable platform in osteochondral therapeutics.While silk has been used as a scaffold for cartilage and/or bone tissue regeneration individually, the materials' ability to support osteochondral tissue regeneration to our knowledge has never been evaluated The human bone marrow stromal cells (hBMSCs) were purchased from Lonza Ltd (USA). All animal studies were carried out under the PI's UK Home Office Project License approval (PPL:40/3361).Bombyx mori (Bm) and Antheraea mylitta (Am) were prepared following the standard protocol described elsewhere 2CO3 for 1 hr to remove completely the highly hydrophilic protein sericin th instar larvae. The silkworms were dissected to obtain the silk glands. The glands were washed thoroughly in de-ionized water to remove the traces of water soluble sericin. The gel like hydrophobic fibroin was squeezed out in SDS-Tris buffer system to dissolve the Am fibroin Aqueous silk protein fibroin solutions of The porous nature of the silk scaffolds was examined using SEM (JEOL JSM 5800) after gold coating. The pore sizes were determined by calculating random 26 pores from SEM image using ImageJ 1.40 g software 1) in a graduated cylinder for 5 min. Hexane permeated through the interconnective pores of the scaffolds causing negligible swelling or shrinkage. After 5 min, the total volume of hexane-impregnated scaffolds with hexane (V2) and only the residual hexane volume (V3) were recorded. The porosity (Є) was calculated as follows: Porosity of the scaffolds was calculated by solvent displacement method using hexane, an inert non-solvent for silk as described elsewhere vitro experiments.Human bone marrow stromal cells (hBMSCs) from hematologically normal donors  were cultivated in α-MEM containing 10% (v/v) FCS. Passage 4 (P4) cells were used for all of the in 5 cells/scaffolds) were seeded dynamically onto silk scaffolds following sterilization as described previously for 24 hrs. and maintained under static culture conditions at 37°C and 5% CO2−8 M dexamethasone, 100 µM ascorbate-2-phosphate and 1× insulin transferring selenium (ITS) −8 M dexamethasone and 100 µM ascorbate-2-phosphate) 2.Equal numbers of BMSCs (2.5×10After 4 weeks for chondroinduced constructs (n = 4) and 8 weeks for osteoinduced constructs (n = 4), cells were labeled with Cell Tracker™ Green 5-chloromethylfluorescein diacetate  and Ethidium homodimer-1  Histological staining was carried out as described elsewhere in vivo studies were carried out under UK Home Office Project License approval (PPL:40/3361).  Osteochondral defects (1.8 mm diameter × 1 mm depth) were created in the patellar groove of the knee joints of male Wistar rats (230–250 g) using a trephine burr. Animals were divided into five groups listed in All After 8 weeks, staining with specific antibodies against type I collagen , and type II collagen  was performed on 5 µm thick formalin-fixed paraffin-embedded tissue. Endogenous peroxidase activity was blocked using 2% hydrogen peroxide and an enzymatic antigen retrieval step was carried out using chymotrypsin (Sigma). Samples were incubated with the primary antibody (overnight at 4°C). Staining was achieved using peroxidase conjugated secondary antibodies  and visualized using an Olympus BX50 microscope. Nuclei were counter stained with hematoxylin.in vitro and in vivo studies. However, report of the use silk fibroin for complete regeneration of osteochondral defects in vivo is sparse Bombyx mori and non-mulberry silk of Antheraea mylitta for in vivo osteochondral repair is novel.The use of silk scaffolds for regeneration of either cartilage or bone has been well documented in previous Am or Bm; pores with more open, interconnected and well defined boundaries were observed in Am, in contrast to Bm. Average pore sizes (based on the measurement of random 26 pores) of Bm and Am 3D porous networks were 72±10 µm (60–96 µm) and 74±10 µm (63–99 µm) respectively. In addition, porosity of the scaffolds ranged between 74 and 82% with a maximum pore size of 82±10 µm in Am scaffolds. The scaffolds were readily equilibrated in culture medium without any need for pre-wetting treatments.Silk fibroin scaffolds prepared by lyophilization  demonstrin vitro (n = 4 for each group) . 3D reconstruction using a series of CLSM slices enabled to observe cell ingrowth and spreading at different depths of the scaffolds. The micrographs visually revealed a high proportion of viable cells (green) in both scaffold groups h group) . After 4h group)  and 8 weh group) , the celin vitro are strongly suggestive of the fact that the hBMSCs had undergone chondrogenesis Alcian blue (AB)/Sirius red (SR) staining revealed a mixed extracellular matrix (ECM) commensurate with fibrous collagen and glycosaminoglycans (GAGs) in all groups . Chondroin vivo experimental constructs re-confirmed the histological findings  indicating closing of the defect. This layer of cells was absent in the empty control (data not shown).A trephine was used to create the 1.8 mm diameter and 1 mm depth osteochondral defects in the patellar groove of the knee joints of Wistar rats . The deffindings  and 9B. findings  and 9A. in vivo experimental constructs for collagens (types I and II), strong staining of type I collagen was observed throughout the ECM of all of the defects with few areas that were non positive .The similar pore sizes of both types of silk scaffolds used in the present study eliminated this as a potential confounder, when investigating the ability of mulberry and non-mulberry silk scaffolds to induce/support osteo/chondrogenesis with the appropriate signaling cues. hBMSCs appeared to preferentially differentiate down the chondrogenic pathway on non-mulberry silk scaffolds whilst preferential osteogenic differentiation of hBMSCs was observed on mulberry silk scaffolds in vivoThe strategic employment of growth factors in the repair of osteochondral defects in vivo constructs examined in this study showed good neo-tissue and host tissue integration, the extent of osteochondral tissue ECM formation not only differed amongst the experimental and control constructs but also between mulberry and non-mulberry silk constructs. All silk scaffold constructs revealed visually indistinguishable dense cellular networks with an intact and visible scaffold lattice. The non-homogenous histological and immunohistochemical staining was indicative of incomplete chondrogenesis in vivo culture. As the aim of the current study was to evaluate the osteochondral reconstruction potentiality of two different silk types, the 8 weeks selected was deemed adequate to interpret the differences based on histology and immunohistochemistry. Substantial differences in the type of ECM formed were observed between the two silk scaffold types (mulberry vs. non-mulberry) and with or without the addition of growth factors. Mulberry scaffold constructs revealed formation of numerous blood vessels enclosed with endothelial cells towards the base while non-mulberry scaffold constructs exhibited fewer blood vessels within the scaffolds and larger chondrocyte like cells at the base indicating possible endochondral ossification. The ECM of mulberry constructs was mainly collagenous (type I collagen in particular) whereas the ECM of non-mulberry constructs was a mix of proteoglycans and collagens (type I and type II collagen). Cumulative findings of the data suggest the ability of silk fibroin to regenerate an osteochondral defect in rat knee joints using a cell free approach with mulberry silk scaffolds differentially favoring osseous tissue formation and non-mulberry silk scaffolds differentially favoring chondral tissue formation after 8 weeks in vivo culture.Whilst all the in vivo is well established in vivo with coaxed osteochondral differentiation may be from complex cues provided in combination with the physical interaction of the construct with specific endogenous niche that help to mediate functional tissue engineering. The results from this study demonstrated the feasibility of using silk scaffolds from two different species as a 3D implantable platform in osteochondral therapeutics.Osteochondral defects are complex to treat due to the nature of two very different tissues involved. Bone is a highly metabolically active and vascular tissue whereas cartilage is an avascular bradytrophic tissue. Silk fibroin's ability to vascularize and home cells in vivo implantation into OCDs, acellular mulberry and non-mulberry silk scaffolds possess an inherent ability to attract endogenous, joint-resident cells capable of differentially differentiating down the osteo/chondral lineages. The main findings of this study was that silk fibroin scaffolds of Antheraea mylitta are more chondroinductive while those of Bombyx mori are more osteoinductive when compared under similar conditions, indicating the potential of using a multi-layered combination of these two different kinds of silk fibroin scaffolds for osteochondral defect regeneration with good integration, the stem cell-silk biomaterial interaction, which is exploited in the context of bone or cartilage promoting niche in this investigation, can also be used for other paradigms of tissue engineering to open up new platforms in regenerative medicine.We believe this to be the first report to show that following


ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation.We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes  Hair follicles produce hair fiber during regular cycles of active growth and relative quiescence HF melanocyte stem cells (MSC) are localized in the outer root sheath (ORS) near the HF “bulge” region, where HF epithelial stem cells reside Numerous factors have been recognized that may affect hair pigmentation, including general metabolism and nutritional status, hair-cycle dependent changes, body distribution, racial and gender differences, hormone-responsiveness, genetic defects and age-associated changes Recent advances in molecular genetics offer some intriguing clues to explain the basis of the diversity in hair pigmentation. Currently, the ‘free radical’ theory is the most popular explanation for the development of non-pigmented hair growth development with age In this study, we used microarrays to determine differences in gene replicate variance between pigmented and non-pigmented HFs. We identified the nucleotide excision repair (NER) pathway may play an important role in modulating MSC attrition and the development of non-pigmented hair growth. Further, we found a key factor of the NER pathway, ERCC3, which may play a key role in maintaining and regulating the fate and behavior of MSCs and mature melanocytes.Gene expression of the tissue groups indicated that 388 genes were differentially expressed between pigmented hair sheaths (HS) and non-pigmented HS with statistical significance . 2289 geWe functionally annotated Several gene function categories were identified with enrichment in significantly differentially expressed genes between pigmented HS and non-pigmented HS , such asGenes in From the “response to radiation” pathway, we selected 11 genes with known functional significance associated with NER for further evaluation by quantitative polymerase chain reaction (qPCR). We also reconfirmed differential gene expression of melanocyte markers, tyrosinase, tyrosinase related protein 1 and tyrosinase related protein 2, to validate our study. The trends, whether for increased or decreased gene expression, were largely, though not entirely, consistent with the microarray data .Because the sensitivity of the qPCR assay is usually higher than that of the microarray assay, discordant results for individual genes could be due to differences in detection limits for the two assays The ERCC3 gene mRNA showed statistically significant downregulation in both non-pigmented HB (0.144 fold) and HS (0.66 fold) by qPCR. This gene, which showed the most consistency in expression trends between the two assays, became a particular focus of interest for further investigation.Dual immunofluorescence labeling was conducted for ERCC3 expression in relation to gp100, a marker for mature melanocytes and premature melanocytes, in terminal scalp HF and nevi tissues. In normal scalp, dual labeled mature melanocyte and ERCC3 positive cells were readily detectable in HF bulb region . Dual laTo examine whether ERCC3 siRNA could selectively knockdown ERCC3 gene expression, we conducted qPCR on transfected melanocytes . Cells tTyrosinase is a rate-limiting enzyme in melanin synthesis. To investigate the effect of ERCC3 on pigmentation, we detected the activity of tyrosinase We compared the global gene expression signature of pigmented and non-pigmented HS and HB by microarray with the aim of detecting differently expressed genes related to pigmentation regulation and white hair. Unsupervised SAM revealed a high degree of similarity in gene expression confirming the homogeneity of the common tissue source for the pigmented and non-pigmented HSs and HBs. Low gene expression variation suggested the heterogeneity of pigmented and non-pigmented HFs is low. Nevertheless, we found some genes with differential expression between pigmented and non-pigmented HF parts. Compared with pigmented HF gene expression profiles, a small group of genes were identified as significantly over-expressed in non-pigmented HFs; on the other hand, a much larger number of significantly differentially expressed genes were identified with a tendency towards decreased expression in non-pigmented HFs compared with pigmented HF gene expression profiles. These results imply that increased dysfunction is the outcome of most molecular processes associated with white HFs.White hair is not simply due to a loss of melanocytes, rather melanocyte function becomes progressively disordered in non-pigmented HFs. Normally amelanotic MSCs in the outer root sheath differentiate and may begin to produce pigment GO analysis of the differentially expressed genes between pigmented and non-pigmented HBs indicated there were significant changes in specific functional gene groups. We found negative regulation of genes in the categories of “melanin metabolic process” (5 genes) and “pigment biosynthetic process” (9 genes) in white HF bulbs consistent with a loss of melanin production in mature melanocytes. We also found significant gene expression changes in the categories of “regulation of cell death” (109 genes) and “positive regulation of cell death” (58 genes) consistent with dysfunctional apoptosis in non-pigmented HBs. This may be more likely to occur in mature melanocytes because HB keratinocytes and fibroblasts reportedly have higher antioxidant enzyme activities than melanocytes 2O2 and other free radicals via the hydroxylation of tyrosine and the oxidation of DOPA to melanin, placing HF melanocytes under high oxidative stress 2O2, even in low doses, can induce cell senescence Incidence of HB melanocyte apoptosis in the aging human HF may be an indicator of oxidative stress induced tissue damage and associated mitochondrial DNA damage. Melanin-synthesis generates HOur results suggest non-pigmented hair could be associated with decreased oxidative phosphorylation coupling efficiency and increased superoxide production in HB melanocytes The outstanding categories, “response to radiation”, “regulation of cellular response to stress” and “regulation of response to stress” were identified with significantly different expression between pigmented HS and non-pigmented HS. These categories include genes which are involved in cell maintenance, gene transcription and DNA repair. Our result is consistent with other studies that suggest radiation-induced stem cell damage can result in non-pigmented hair Mature melanocytes and MSCs are under different oxidative stress conditions, but the same result may ensue; cellular DNA damage, gene transcription defects, and subsequent progressive dysfunction. DNA injury may compromise gene transcription and cell replication and eventually cause cellular senescence (growth arrest) or cell death (apoptosis) Our results showed ERCC3 was statistically significantly down-regulated in white HF. ERCC3 along with ERCC2 are components of the transcription factor IIH (TFIIH) which is involved in gene transcription initiation. The TFIIH complex also plays a key role in NER by opening damaged duplex DNA at the damage site through the action of its two helicases, ERCC2 and ERCC3. Mutation in either ERCC3 or ERCC2 can cause instability of the whole TFIIH complex Insufficient ERCC3 function reduces DNA repair which correlates with an increased cellular sensitivity to oxidative DNA damage, cell death, and consequent depletion of cell renewal capacity similar to ERCC2 deficiency Insufficient ERCC3 function may directly reduce the ability to transcribe genes In conclusion, our data suggest that primary transcription decay is likely related to the process of pigmentation loss in hair. An imbalance between DNA damage and DNA repair transcription capacity may accelerate physiological “hair graying”. We suggest the “aging” of melanocytes due to oxidative stress sensitivity affects hair pigmentation. We propose that reduced ERCC3 activity in HF melanocytes may also reduce transcriptional sufficiency leading to reduced melanogenesis activity. Stochastic damage may also accumulate in MSCs and mature HF melanocytes because of insufficient NER gene function. In the longer term, insufficient NER function may cause cell senescence and death in MSCs and mature melanocytes might also have a reduced life span. Strengthening DNA repair transcription and tapping into this NER pathway, especially ERCC3 may slow down the process of pigmentation loss in hair.Human HFs were collected from 4 mm scalp biopsies of healthy individuals who presented with a combination of pigmented and non-pigmented white hair . Using a common source for the comparison groups negated any potential differences in gene expression due to differences between individuals in terms of environmental exposure or systemic hormonal activity. The samples were derived from 7 females 43–55 years of age (mean of 49.8 years±SD 4.56 years). All samples were provided through the Department of Surgery and the Department of Dermatology and Skin Science, University of British Columbia, with approval from the University Clinical Research Ethics Board. All participants gave written informed consent before participation in the study.HF (n = 10−20 per sample group per subject) were microdissected partly using the method of Philpott and colleagues Anagen stage HF were separated into two sample groups; those with pigmentation, where the dermal papilla was hidden in a strongly pigmented HB and pigment-activity was evident in the hair shaft; and those where macroscopically no pigmented melanocytes could be detected in the HB and the hair shaft appeared fully unpigmented. HFs with apparent pigmentation dilution, but not fully devoid of pigmentation , were veTotal RNA was isolated from microdissected tissue with an RNeasy Fibrous Tissue Midi Kit  according to the manufacturer’s protocols. RNA quality and the quantity were measured using the Agilent 2100 bioanalyzer . Human Operon v.2.1 (21 K) glass arrays were produced  by the Microarray Facility of the Prostate Centre at Vancouver General Hospital, Vancouver, Canada http://www.ncbi.nlm.nih.gov/geo/) encompassed by the series number (GSE24009). The raw data were inputted into Genespring 7.2 program  and analyzed using protocols detailed elsewhere q-value and a 2 fold cut-off  database ( cut-off . The cutwww.geneontology.org), identified “enriched” or “depleted” gene function categories Functional classification of genes using DAVID software, based on the Gene Ontology (GO) database   and melanocyte marker, gp100 . The pre-melanosomal marker gp100 in vitro study. Cells were maintained in Medium 254 supplemented with human melanocyte growth supplement (Invitrogen). Cells were plated at a density of 2.5 to 5×105 cells/ml. Using PromoFectin-siRNA transfection reagent , cells were transfected with either ON-TARGETplus Non-Targeting Pool (50 nM)  or ON-TARGET plus Smart pool ERCC3 siRNA (50 nM) ; 24 hours after transfection, cells were collected. Cell numbers were counted before and after transfection.Although, there are some behavioral differences between adult and neonatal melanocytes in culture, there are no age-dependent differences in melanin content and cell proliferation rates between neonatal and adult cultured melanocytes. Neonatal melanocytes are considered an acceptable model for studying the mechanisms of adult-onset hyperpigmentary disorders and melanogenesis Tyrosinase activity was spectrophotometrically assayed as DOPA oxidase activity using a previously reported method Table S1Gene transcripts with significant upregulation (a) and downregulation (b) in non-pigmented hair bulb versus pigmented hair bulb. Top twenty five genes sorted by the false discovery rate .(DOC)Click here for additional data file.Table S2Gene transcripts with significant upregulation (a) and downregulation (b) in non-pigmented hair bulb versus pigmented hair bulb. Top twenty five genes sorted by the false discovery rate .(DOC)Click here for additional data file.Table S3Differences in Gene Ontology categorization analysis between non-pigmented hair bulb versus pigmented hair bulb at GO level 3. All categories identified are shown.(DOC)Click here for additional data file.Table S4Differences in Gene Ontology categorization analysis between non-pigmented hair sheath versus pigmented hair sheath at GO level 3. All categories identified are shown.(DOC)Click here for additional data file.Table S5Primer sequences for defined genes.(DOC)Click here for additional data file.


Relentless selective pressures exerted by viruses trigger arms race dynamics that shape the evolution of even critical host genes like those involved in iron homeostasis. TFR1 successfully balance the opposing selective pressures exerted by host and virus. We find that while the sequence of rodent TfR1 is generally conserved, a small set of TfR1 residue positions has evolved rapidly over the speciation of rodents. Remarkably, all of these residues correspond to the two virus binding surfaces of TfR1. We show that naturally occurring mutations at these positions block virus entry while simultaneously preserving iron-uptake functionalities, both in rodent and human TfR1. Thus, by constantly replacing the amino acids encoded at just a few residue positions, TFR1 divorces adaptation to ever-changing viruses from preservation of key cellular functions. These dynamics have driven genetic divergence at the TFR1 locus that now enforces species-specific barriers to virus transmission, limiting both the cross-species and zoonotic transmission of these viruses.Transferrin Receptor (TfR1) is the cell-surface receptor that regulates iron uptake into cells, a process that is fundamental to life. However, TfR1 also facilitates the cellular entry of multiple mammalian viruses. We use evolutionary and functional analyses of TfR1 in the rodent clade, where two families of viruses bind this receptor, to mechanistically dissect how essential housekeeping genes like  Genetic differences between mammalian species dictate the patterns of viral infection observed in nature. They also define how viruses must evolve in order to infect new mammalian hosts, giving rise to new and sometimes pandemic diseases. Because viruses must enter cells before they can replicate, new diseases often emerge when existing viruses evolve the ability to bind to the cell-surface receptor of a new species. At the same time, host cell receptors also evolve to counteract virus attacks. This back-and-forth evolution between virus and host can lead to an arms race that shapes the sequences of the proteins involved. In wild rodent populations, the retrovirus MMTV and New World arenaviruses both exploit Transferrin Receptor 1 (TfR1) to enter the cells of their hosts. Here we show that the physical interactions between these viruses and TfR1 have triggered evolutionary arms race dynamics that have directly modified the sequence of TfR1 and at least one of the viruses involved. Computational evolutionary analysis allowed us to identify specific residues in TfR1 that define patterns of viral infection in nature. The approach presented here can theoretically be applied to the study of any virus, through analysis of host genes known to be key to controlling viral infection. As such, this approach can expand our understanding of how viruses emerge from wildlife reservoirs, and how they drive the evolution of host genes. Transferrin receptor (TfR1) is the cell-surface receptor for iron-loaded transferrin circulating in the blood Muridae rodents specifically of the genus Mus, including Mus musculus, the house mouse  are host proteins like RIG-I and TRIM5α that recognize and destroy viruses that are attempting to replicate inside of host cells TFR1 and demonstrate that both the protein sequence and the interaction specificities of this receptor are far from static. Using a small evolutionary dataset consisting of TFR1 gene sequences from only seven rodent species, we identify specific codons in TFR1 that have been repeatedly targeted by positive selection for amino acid replacement. We find that these rapidly evolving positions correlate to the surfaces on TfR1 that mediate interaction with the two rodent viruses that bind this receptor. We demonstrate experimentally that mutations at these specific receptor residues are potent at altering interactions with virions while not altering receptor expression or function. We show that this evolutionary scenario has driven genetic divergence at this receptor locus that now enforces species barriers to viral transmission. We address the implications of these findings for human TfR1 and identify a human SNP that conveys some protection against cellular entry of a zoonotic rodent arenavirus. Our study demonstrates that the influence of viral pathogens on mammalian genomes goes well beyond the shaping of antiviral genes, as we can now appreciate that even the sequence of important housekeeping genes can be shaped by unremitting antagonism by viruses. However, in this case, collateral damage to cellular functions must be carefully controlled as the evolutionary battle with viruses plays out.Arms races have not traditionally been documented in important housekeeping genes. Here, we document recurrent positive selection in rodent TFR1 in rodents, where two different virus families use this receptor for cellular entry. The type of selective pressure that has acted on a gene can be inferred from the pattern of mutations that it has accumulated over time TFR1 from seven rodent species, five of which are known host species for the New World arenaviruses or MMTV  fit the data significantly better than models where positive selection is not allowed . Like all virus surface proteins, GP1 will have also experienced selection for immune escape, a complication that makes signatures of dN/dS>1 more difficult to interpret in viral genes than in host genes. However, GP1 residues in direct contact with TfR1 are unlikely to successfully mutate for the purpose of immune escape during an active infection. An arms race between rodent TfR1 and arenavirus GP1 is thus supported by the rapid evolution of each partner in this interaction, specifically in residues that are known to mediate contact with the other.Arms races are predicted to drive positive selection in both the host and virus genes involved, so we next analyzed the gene encoding the arenavirus surface protein, GP, for signatures of positive selection. Because the co-crystal structure has been solved of the Machupo virus surface glycoprotein subunit GP1 in complex with TfR1, the specific residues on GP1 that interact with TfR1 are known  and the North American deer mouse (Peromyscus maniculatus)  . The fulCricetidae species are highly recalcitrant to entry by MMTV  led to a significant increase in MMTV entry  and HEL299 (lung). Lung cells are especially relevant since arenaviruses are transmitted to humans through respiratory inhalation. In both cases, expression of the minor 212V allele was again protective against virus entry compared to the wild-type allele , while TfR1-utilizing arenaviruses have only been found in the Americas. We sequenced TFR1 represents the first case, to our knowledge, where the evolution of a single host gene is driven by two host-virus arms races at once. In the case of the MMTV binding surface, this has played out through three residue positions coordinated in three-dimensional space. In the arenavirus binding surface, the target of selection has been a small surface-exposed structural feature, in which we were able to detect positive selection of three of the residues. Outside of rodents, TfR1 is used by a third family of viruses, the parvoviruses, and carnivore TFR1 is also under positive selection TFR1 evolution has thus been shaped by viruses in two separate species groups (rodents and carnivores) and by every viral pathogen known to use this receptor. These findings now explain how TFR1 became divergent enough to create species-specific interactions with all three of these virus families. If even a few residue positions can evolve to block virus entry without collateral damage to cellular function, host-virus arms race dynamics can unfold even in genes encoding highly conserved and essential housekeeping proteins.In this study we show that the protein sequence and interaction specificities of rodent TfR1 have been dynamic over time, shaped by selective pressures imposed by viruses. These dynamics have played out through mutations accumulated at just a small number of residue sites, where mutations decrease virus entry without measurably affecting receptor expression or iron-transport functions. TFR1 can be put into contrast with other types of pathogen-driven positive selection of host genes. The human CCR5 gene encodes a co-receptor for HIV cellular entry. Some humans encode a variant allele of CCR5, CCR5Δ32, where a 32 base pair deletion gives rise to a defective receptor that is not expressed on the cell surface TFR1 L212V, CCR5Δ32 pre-dates HIV and may or may not have had any functional significance before the HIV pandemic. Nonetheless, it has become highly relevant in a world with HIV/AIDS. Like HIV, most simian immunodeficiency virus (SIV) strains also use CCR5 as a co-receptor. In a fascinating case of convergent evolution, some sooty mangabeys and red-capped mangabeys also encode null or defective alleles of CCR5DARC gene encodes a chemokine receptor that is used as an entry receptor by some malaria-causing Plasmodium species. A cis-regulatory polymorphism that silences DARC expression in erythrocytes has arisen independently in human populations from different parts of the world and is highly protective against Plasmodium vivax and Plasmodium knowlesi infection DARC in African baboons, and these are associated with resistance to a malaria-like parasite common in baboon populations This evolution of CCR5 and DARC examples represent a more common mode of pathogen-driven positive selection (not recurrent) than the one demonstrated for TFR1, and there are several important differences. When receptor genes experience hypomorphic mutations, the predominant evolutionary strategy available to viruses will be to use a new receptor altogether. Indeed, the SIV strains that infect sooty and red-capped mangabeys (SIVsmm and SIVrcm) have both evolved to use alternate co-receptors CCR5Δ32 homozygous humans have also been reported to be infected with HIV, again through mutations that allow the virus to use an alternate co-receptor (CXCR4 in this case). Hypomorphic mutations in receptors are not expected to be “serially replaced” due to arms race dynamics. Rather, viral evolution to use a new receptor ends the arms race with the original receptor gene and starts a new one with the new receptor gene. The CCR5 and DARC examples also involve evolutionary time scales millions of years shorter than what has been demonstrated in the current study; because these hypomorphic alleles are circulating in populations of individuals and are not shared between species, they have arisen relatively recently. Also, because these mutations simply reduce cellular expression of the encoded receptors, they presumably have some negative fitness effect on the host. The TfR1 example that we provide here is unique because solutions to viral entry have been found that appear to lack collateral damage to transferrin binding, and presumably to other host functions as well. Because of this, these mutations become common or fixed in populations where they occur, and are serially replaced as viruses continue to evolve and as rodents continue to speciate.These XPR1 gene encoded by rodents of the genus Mus, each with its own pattern of virus susceptibilities. The viruses that use this receptor are also highly variable in the receptor-binding portion of their surface protein, Env. High levels of sequence divergence and disparate interaction specificities have also been observed between the entry receptor TVB encoded by birds and the avian leukosis virus (ALV) strains that use this receptor There is reason to believe that host-virus arms races are also shaping the protein sequence of other virus entry receptors in the manner described here. There are several other examples where significant sequence and functional divergence exist both on the side of a virus and its host entry receptor. For instance, certain strains of murine leukemia virus (MLV) use the rodent XPR1 receptor for cellular entry Traditionally, TfR1 has been viewed as a housekeeping protein with an immensely important and conserved role in the cell. This study provides a much richer understanding of the multiple dynamic roles that this receptor is balancing in nature.TFR1 and Machupo gp1 sequences were analyzed for positive selection. Database accession numbers for sequences used are listed in TFR1 gene tree matches the species tree of these rodents gp1 sequences represent viral isolates from the same population, GARD gp1 alignment to confirm the lack of phylogenetic breakpoints indicative of recombination. For both datasets, maximum likelihood analysis of dN/dS was then performed with codeml in the PAML 4.1 http://www.pdb.org/) and rendered using PyMOL (http://www.pymol.org).Rodent M. musculus (mm9) R. norvegicus (rn4) pol as a query. Both of these approaches identified the Peromyscus maniculatus ERV buried in the sequence of GenBank record EU204642 (a BAC clone containing the deer mouse beta-globin gene cluster). A relatively young age of this ERV can be inferred from the fact that one open reading frame (pol) is still uninterrupted, and from the observation that the 5′ and 3′ LTRs differ at only 1 out of 917 positions. The giraffe, bison, and musk ox sequences are from Full-length MMTV sequences were obtained on GenBank . These sequences were used to BLAT TFR1 (rs41301381) was identified in data deposited by the 1000 Genomes Project (http://browser.1000genomes.org). As of Release 12, L212V had been found as a heterozygous SNP in 11 individuals, with no homozygous carriers identified. Three of these individuals were Han Chinese from the South (CHS population), six were Han Chinese from Beijing (CHB population), and two were Japanese individuals (JPT population). In total, 11 out of 286 Asian individuals surveyed were heterozygous at this position, yielding a genotypic frequency of 0.038 in Asia. This SNP has not been included in the HapMap Genotyping Project (as of Release 28).The L212V SNP in human −1 penicillin, 100 µg ml−1 streptomycin, and 2 mM L-glutamine (Cellgro). Human, Mus musculus, Calomys musculinus, Calomys callosus, and Zygodontomys brevicauda TFR1 with an encoded C-terminal FLAG tag were moved from pcDNA3.1 (+) vectors (described previously TfR1 for TA cloning: 5′-TTAATACGACTCACTATAGGG-3′ and 5′-TAGAAGGCACAGTCGAGGC-3′. Gateway LR recombination (Invitrogen) was performed to transfer TFR1 genes from pCR8 into the entry site in a Gateway-converted LPCX retroviral vector. Site-directed mutagenesis of the human, M. musculus, and Z. brevicauda TFR1 orthologs was performed using QuikChange Site-Directed Mutagenesis kit (Stratagene). Plasmids encoding Machupo, Junin, and Guanarito GP have been described previously Human embryonic kidney 293T cells (ATCC CRL-11268), HEK293 cells (ATCC CRL-1573), human embryonic lung HEL299 cells (ATCC CCL-137), and canine kidney MDCK.2 cells (ATCC CRL-2936) were all maintained in Dulbecco modified Eagle's medium (Cellgro) supplemented with 10% fetal bovine serum (Gibco), 100 units mlTFR1 retroviral vectors were packaged in 293T cells by co-transfecting them along with the NB-MLV packaging plasmid pCS2-mGP −1 puromycin was added to select for transduced cells (1.0 µg ml−1 puromycin was added when creating the HEK293 and HEL299 stable cell lines). These receptors have a C-terminal FLAG tag that is extracellular when the receptor is at the cell surface TFR1 alleles were made in MDCK, HEK293, and HEL299 cells as described above.The above described LPCX:5 cells per well in a 24-well plate and, after 24 h, infected with pseudotyped virus along with 5 µg ml−1 polybrene. The plates were spinoculated with centrifugation at 350g for 1.25 h at 30°C. After 2 h of incubation at 37°C, cells were washed once with PBS and the media was replaced. Two days postinfection, cells were analyzed by flow cytometry. Cells were first gated for live cells and then, using an anti-FLAG antibody conjugated with Allophycocyanin , further gated such that all samples were narrowed to the same log decade of receptor expression (capturing the majority of cells but excluding outliers). Where TfR1 expression levels are reported, this is the mean fluorescent intensity within this gated population . These same 10,000 cells were scored for expression of GFP . Analysis of flow cytometry data was performed using FlowJo 8.8.6 .Arenavirus GP or MMTV Env pseudotyped MLV recombinant retroviruses were packaged in 293T cells. Fugene (Roche) was used to co-transfect the GFP-encoding transfer vector pQCXIX (BD Biosciences) along with plasmids encoding MLV Gag-Pol and one of the viral surface glycoproteins Machupo GP, Junin GP, Guanarito GP, or MMTV Env. After 48 h, supernatants containing viruses were harvested, filtered, and frozen at −80°C. For entry assays, cell lines stably expressing various TfR1 orthologs or human alleles were plated at a concentration of 1.0×105 cells/tube. The cells were washed with DPBS with 1% ovalbumin (Sigma). The cells were then resuspended in 200 µL of DPBS with 1% ovalbumin containing 1∶500 dilution of FITC-conjugated Mouse transferrin  and incubated at 37°C for 60 min. Anti-DDDDK (FLAG) tag antibody conjugated with Allophycocyanin  was added to the cells at a 1∶100 dilution and incubated on ice for 20 min. The cells were then washed twice, resuspended in DPBS with 1% ovalbumin, and then analyzed by flow cytometry. Cells were first gated for live cells and then further gated such that all samples were narrowed to the same log decade of receptor expression (capturing the majority of cells but excluding outliers). Where TfR1 expression levels are reported, this is the mean fluorescent intensity within this gated population . These same 10,000 cells were simultaneously analyzed for transferrin binding with FITC. Analysis of flow cytometry data was performed using FlowJo 8.8.6 .MDCK.2 stable cell lines expressing FLAG-tagged TfR1 orthologs were trypsinized and aliquoted in triplicate at a concentration of 2.5×10Figure S1pro-pol or (B) for select viruses where full-length sequence was available, approximately 5,000 aligned bases spanning from the middle of gag to the end of pol. In bold are exogenous viral sequences. All others are endogenous viral sequences found integrated in the genomes of the indicated host species. In both panels, maximum likelihood trees are shown. On each node are bootstrap values, given as percentage of 1,000 replicates. Trees were rooted with FIV (feline immunodeficiency virus), a lentivirus that is not in the beta-retrovirus family. The two human ERVs included here (HERV-K50B and HERV-K33) are the highest scoring HERV matches to MMTV, based on BLAST search scores.Phylogenetic analysis of MMTV-like beta-retroviruses. Beta-retrovirus phylogeny constructed from (A) approximately 900 bases in the region of (PDF)Click here for additional data file.Figure S2Peromyscus maniculatus genome. The TfR1 binding determinants of MMTV Env have not fully been mapped, but a TfR1-binding motif has been described Evolution of the putative receptor binding motif of MMTV Env. A partial alignment of the viral protein Env is shown. The alignment includes all available rodent MMTV and MMTV-like virus sequences, as described in more detail in the manuscript, including the endogenous retrovirus found in the (PDF)Click here for additional data file.Table S1TFR1 sequences. This table summarizes the codon-based analysis of dN/dS performed on rodent TFR1 sequences.PAML analysis of rodent (PDF)Click here for additional data file.Table S2gp1 sequences. This table summarizes the codon-based analysis of dN/dS performed on Machupo virus gp1 sequences.PAML analysis of Machupo virus (PDF)Click here for additional data file.