Various mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive (“lytic”) and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored. Herpes simplex virus 1 (HSV-1) switches between two infection programs, productive (“lytic”) and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency. Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that establishes lifelong latency in sensory neurons following productive (“lytic”) infection in peripheral tissues (reviewed in reference 2–9–13–miRNAs are derived from transcripts (pri-miRNAs) with embedded stem-loops. Typically, Drosha, a nuclear RNase, cleaves pri-miRNAs into smaller hairpin-shaped RNAs known as pre-miRNAs. Exportin 5 then binds pre-miRNAs in a Ran-GTP-dependent manner and exports them into the cytoplasm, where they are further cleaved by another RNase, Dicer, releasing small RNA duplexes. One strand of each duplex is subsequently loaded into an RNA-induced silencing complex containing the Argonaute (Ago) protein, which targets the complex to complementary mRNAs  analyses, which provide no information regarding miRNA precursors. In contrast, Northern blot hybridization (Northern) permits detection of both pre-miRNA and mature miRNAs with high specificity. Starting with the initial reports of HSV miRNAs , 24, 25,To understand this phenomenon, we conducted a series of Northern-based experiments. The results not only revealed distinct HSV-1 miRNA expression patterns in cell culture and mouse ganglia but also uncovered a regulatory mechanism that inhibits pre-miRNA nuclear export.LAT promoter does not affect their expression during lytic infection as it does during latency)  cells, we analyzed expression of miR-H1 to miR-H6 using two additional cell lines, human embryonic kidney 293T) cells and mouse neuroblastoma (Neuro-2a) cells, following infection at multiplicities of infection (MOI) of 10 and 30, respectively, by HSV-1 wild-type (WT) strain KOS. These miRNAs (and the corresponding pre-miRNAs) are expressed as late gene products in Vero cells from unknown promoters (deletion of the latency) . In addilatency) . All 6 platency) . Mature latency) , and eve93T cellslatency) , 12. Thu10.1128/mBio.02856-18.1FIG S15 PFU of HSV-1 strain KOS per eye. At various days postinfection (dpi), trigeminal ganglia were collected. At 30 dpi, some ganglia were explanted and cultivated for various days postexplantation (dpe) before the ganglia were harvested for RNA. Sixteen ganglia were pooled for each condition, and small RNAs (<200 bases) were isolated and analyzed by Northern blot hybridization. RNAs purified from 293T cells that had been mock infected or infected for 18 h were run alongside (see last two lanes) the RNAs from ganglia to provide markers. The sources of RNAs and time points are indicated at the tops of each series (left or right) of Northern images. Positions of miRNA species are indicated to the right of the images. Ethidium bromide signals at ∼80 bases from the gels used for each series of Northern images are displayed at the bottom. An independent experiment is shown in FIG S1, PDF file, 1.3 MB.HSV-1 pre-miRNA and mature miRNA expression in trigeminal ganglia at different times after infection or after reactivation from latency. Mice were mock infected or infected on the cornea with 2 × 10Copyright © 2019 Pan et al.2019Pan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo, we performed two independent experiments  by Northern blotting. We also explanted some ganglia at 30 dpi and cultivated them for 1 to 3 days to reactivate the virus before collecting the tissues for RNA analysis. miR-H2, miR-H4, and miR-H5 showed one expression pattern: Their mature miRNAs were readily detected after 6 dpi, with levels steadily increasing through 30 dpi and then decreasing during reactivation . We then produced lentiviruses and used them to stably transduce 293T and Neuro-2a cells. We monitored miRNA expression following doxycycline (Dox) addition to the transduced cells. The three miRNAs differed greatly in their final pre/mature ratios, with miR-H4 having the lowest and miR-H3 the highest in both the 293T-derived and Neuro-2a-derived cell lines , suggestTwo hypotheses could explain the high pre/mature miRNA ratios: (i) block of pre-to-mature conversion and (ii) expedited degradation of mature miRNA. To investigate these hypotheses, we analyzed miRNA stability following addition of a transcription inhibitor, actinomycin D (ActD). In transduced 293T cells, at 3 days following Dox addition, mature miR-H2, miR-H3, and miR-H4 were all stable for 8 h following ActD addition, but the corresponding pre-miRNAs were all much less stable  and B. I10.1128/mBio.02856-18.2FIG S2FIG S2, PDF file, 0.4 MB.Stability of pre-miRNAs and miRNAs in HSV-1-infected cells. 293T cells were infected with HSV-1 strain KOS (MOI = 10). At 2 (left panel) or 8 (right panel) hpi, 1 μg/ml ActD was added, and cells were harvested at the times indicated at the top of the images. miR-H4 was analyzed by Northern blot hybridization. Results of experiments examining miR-H2 are shown in Copyright © 2019 Pan et al.2019Pan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Us11 and Us12 open reading frames such that miR-138 would be encoded within the Us12 3′ untranslated region (UTR) and Us11 5′ UTR and thus would be expressed from both the Us12 immediate early and Us11 late promoters (Us11 has little if any impact on miRNA biogenesis (see below). As expected for transcription from both immediate early and late promoters, pre-miR-138-1 expression from WTLyt138 virus appeared to be biphasic, with initial expression at 2 hpi followed by a decrease in the level of expression at 5 hpi and then an increase again after 5 hpi  or for separation into nuclear and cytoplasmic fractions prior to RNA purification. The resulting RNAs were subjected to Northern blot hybridization, probing for the RNAs indicated to the right of the panels. Mock infection times and times postinfection are indicated at the top of the panel. The fractions are indicated above the images. T, total RNA; N, nuclear fraction; C, cytoplasmic fraction. This experiment was performed multiple times. Similar results were obtained in an experiment using WTLyt138 virus . DownloaCopyright © 2019 Pan et al.2019Pan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Us11 start codon, showed pre/mature ratios for miR-H1 and miR-H2 similar to those of the corresponding rescued virus (data not shown). Because we detected a weak immunoreactive band that comigrated with strain Patton Us11 in pAUs11-infected cells on a Western blot , and the levels of pre-miR-H6 were similar in the nuclear and cytoplasmic fractions, which were cleanly separated based on the U1 and lys-tRNA markers. Taken together, these results show that ICP27 contributes to high pre/mature ratios and to retention of pre-miRNA in the nucleus.Another candidate for impeding miRNA biogenesis is ICP27, which plays a role in mRNA nuclear export , an inhibitor of HSV DNA synthesis. At 8 hpi, infected cells were either mock treated or treated with ActD, and RNA was harvested immediately or at 14 hpi. In the absence of ACV and ActD, the miR-138 pre/mature ratios increased between 8 and 14 hpi  to E. AdBy characterizing HSV-1 pre-miRNA and miRNA expression in transduced and infected cells and in mouse ganglia, we found that although these miRNAs differed in inherent biogenesis efficiencies, conversion of pre-miRNA to miRNA was considerably less efficient during lytic infection than during latent infection or following lentiviral transduction, regardless of the miRNAs or cell types tested. Much if not all of this impairment of miRNA biogenesis was due to a block in pre-miRNA export from the nucleus to the cytoplasm. High pre/mature ratios occurred late in infection and were dependent on ICP27 and viral DNA synthesis but not on Us11. Below, we discuss these results and their implications for HSV-1 infection.Our Northern results show that all HSV-1 miRNAs tested exhibited high pre/mature ratios during lytic infection of multiple cell lines. Consistent with this, most exhibited high pre/mature ratios in ganglia at 3 dpi, arguing for a regulatory mechanism that applies to both acute ganglionic infection and lytic infection of cultured cells. During latency, pre/mature ratios were considerably reduced, arguing that miRNA biogenesis became more efficient, presumably due to reduced expression of the viral function(s) that impaired miRNA biogenesis. This mechanism then likely contributed to high expression of these miRNAs during latency.Based on our results from lentivirus-transduced cells, the order of intrinsic efficiencies of conversion of pre-miRNA to miRNA is miR-H4 > miR-H2 > miR-H3, consistent with the relative pre/mature ratios for these miRNAs in infected cells and ganglia and suggesting that intrinsic biogenesis efficiencies contribute to determining expression levels. The finding of different intrinsic biogenesis efficiencies explains the inability to detect mature miR-H3 during lytic or latent infection by Northern analysis and the inability to detect pre-miR-H4 in infected ganglia. Variability in intrinsic biogenesis efficiency may reflect sequence-specific differences in pre-miRNA export and/or Dicer cleavage , 39. RegmiRNA biogenesis during lytic infection of cells is temporally regulated. When detectable at early times of infection , pre-miRNAs convert efficiently to miRNAs, but the level of conversion is reduced later in infection. Moreover, pre-miRNAs exhibit short half-lives in the presence of ActD at 2 hpi but are much more stable at 8 hpi. Thus, impairment of miRNA biogenesis is a late infection event. Consistent with this, ACV treatment resulted in both reduced pre/mature ratios and reduced stability of pre-miRNA in the presence of ActD. Similarly, our finding that pre-miRNAs were depleted in the cytoplasm and enriched in the nucleus at 12 hpi, but not at 5 hpi, indicates that inhibition of nuclear export of pre-miRNAs occurs late. Thus, the results strongly suggest that inhibition of nuclear export of pre-miRNAs explains the impairment of miRNA biogenesis late during lytic infection. However, we cannot exclude the possibility of effects on other steps. Blocking nuclear export of pre-miRNAs is likely not unique to HSV-1 infection. Baculovirus encodes a miRNA that was reported to target the Exportin-5 cofactor Ran . AccumulICP27 also greatly reduced pre/mature ratios for HSV-1 miRNAs. Interestingly, this deletion reduced the expression of the pre-miRNAs with no reduction or possibly an increase in the levels of the mature miRNAs, which we suggest was due to these pre-miRNAs being late viral gene products  to combat an RNAi-mediated antiviral response, although the functional relevance of this suppressive activity in mammals remains to be established . Mammali55–60–Kan) flanked by one I-SceI site was then cloned into the EcoRI site of the resulting plasmid using the following two primers: primer CTTAGAATTCGGATCATCAATCATCTGCCTTCCCAGGACCACCCAGCAGTGTTGCACGTATAGGGATAACAGGGTAATCGATTT and primer CTTAGAATTCGCCAGTGTTACAACCAATTAACC. The resulting plasmid was used as a template to amplify a cassette to be recombined with the HSV-1 genome using the following two primers: primer AATCCGGTAACCCGTTGAGTCCCGGGTACGACCATCACCCGAGTCTCTGGAATTCGGATCATCAATCATCTGCC and primer GGGTTGGGTCTGGCTCATCTCGAGAGACACGGGGGGGAACCACCCTCCGCGTAGGGTGGGATAATGGGGTGG. The subsequent recombination of this cassette with the viral genome and removal of Kan were conducted as described previously . Cells were infected by adding virus at the indicated MOI, incubating for 1 h at 37°C with gentle shaking every 15 min for adsorption, and replacing the supernatant with fresh media and incubating at 37°C for the indicated times.32P]ATP (Perkin Elmer) in a total volume of 25 μl for 1 h at 37°C before this mixture was added into the hybridization tube. The subsequent hybridization and washing conditions were the same as those used with the nonradioactive method. After the last wash, the membrane was exposed to a phosphor screen and visualized by a phosphorimager. Band intensities were quantified by Quantity One 1-D Analysis Software (Bio-Rad). The probe sequences and optimized hybridization and washing temperatures for different RNAs are listed in RNAs of <200 nucleotides were purified from cultured cells or pooled mouse ganglia using a miRNeasy minikit (Qiagen) following the manufacturer’s instructions and were resolved on Novex 15% Tris-borate-EDTA-urea polyacrylamide gels (Thermo Fisher). We used radioactive and nonradioactive methods interchangeably for Northern blot hybridization. The nonradioactive method followed a previously described protocol  (7 weeks old) were infected with HSV-1 strain KOS as described previously . At the The synthesized gBlocks gene fragment described above was used as a miR-138-expressing sequence. miR-H2-expressing and miR-H3/H4-expressing sequences were amplified from HSV-1 WT BAC using primers TGACCTCGAGTTTTCCTGGCCCGACCCGCGCCTCTT and CTGTGAATTCGACCCCATAGTGATCAGCGACTCC for miR-H2 and primers TGACCTCGAGGACGCGGACTCGGGAACGTGGA and CTGTGAATTCTCGCGCGCGGCCCTTTAAAGGCG for miR-H3/H4. Each miRNA-expressing sequence was inserted between the XhoI and EcoRI restriction sites of pTRIPZ  (a generWestern blotting was performed as described previously . The folCell fractionation was performed using a tissue grinder (Wheaton) (1 ml) as previously described . RNA was


CD3D, CD3E, and CD3Z gene defects cause severe combined T- and B-cell immunodeficiency. However, CD3G mutations alone lead to a less severe condition, which is mainly characterized by autoimmunity. In the present study, we report the case of a 36-year-old male who presented with recurrent sinopulmonary infections without opportunistic infections; this was compatible with hypogammaglobulinemia, but normal PHA-lymphocyte proliferation. This patient had the common variable immunodeficiency (CVID) phenotype and received regular immunoglobulin infusions over 20-years; he gradually developed nodular regenerative hyperplasia over a 5-year period. Distinct from the previously reported CD3G mutations, which mainly present as autoimmunity, the novel CD3G deletion (c.del213A) in our patient caused an obvious decrease in switched memory B cells and diminished CD40L expression. However, sufficient Treg suppression function was maintained so that he remained free of autoimmune thyroiditis (AIT), inflammatory bowel disease (IBD), and autoimmune pancytopenia. A PubMed search for this rare disease entity revealed seven Turkish and two Spanish patients (five unrelated families). Among a total of 20 alleles, there were 14 splicing mutations (80(-1)G>C), two missense mutations (c.1G>A), two nonsense mutations (c.250A>T), and two deletions (c.del213A). Three patients presented with isolated AIT without significant infections. Three patients died, one from a severe infection at 31 months, one from post-transplant respiratory failure due to viral pneumonia at 17 months, and one from graft-vs.-host disease at 47 months. Those experiencing opportunistic infections, severe life-threatening infections in need of hematopoietic stem cell transplantation, and IBD-like diarrhea had a significantly higher mortality rate compared with those without these features . The patients with AIT had a significantly better prognosis (p = 0.0124) to those without AIT. Our patient with the novel CD3G mutation presented with predominant B-cell deficiency overlapping with the CVID phenotype but without recognizable autoimmunity, which was consistent with his normal Treg suppression function.The T-cell receptor (TCR)/CD3 complex is crucial for T-cell development and regulation. In humans,  The integrity of the T-cell receptor/CD3 (TCR/CD3) complex orchestrates T-cell maturation and activation. Before the TCR/CD3 complex reaches the membrane, TCRα/β heterodimers associate with three invariant dimers , which comprise the CD3 complex . After iThe complete absence of CD3δ or CD3ε chain expression has been shown to block the development of TCRα/β T cells in both humans and mice , and humCD3G deletion mutation. The CD3γ protein it coded for was truncated and lacked the ITAM domain, but global Treg cell function was unexpectedly maintained. To the best of our knowledge, this was the first patient of Chinese ethnicity to be identified with this novel CD3G mutation. In order to optimize his clinical management, a comprehensive review of previous phenotypes, genotypes, treatments, and prognoses was conducted by searching PubMed  at 15-years old and received regular immunoglobulin infusions over the next 20-years. Whole exome sequencing (WES) was performed and revealed a novel g PubMed –13. The Prior to their inclusion, the patient and healthy control provided written informed consent for the collection and publication of their data within the present study. All human samples were obtained using protocols approved by the Institutional Review Board at Chang Gung Memorial Hospital (protocols 201601893A3 and 104-9578A3) and met the Institutional Review Board standards for the ethical conduct of research with human subjects in accordance with the Declaration of Helsinki.), IgD-PE (clone IA6-2), CD27-APC (clone M-/t271), CD45RO-PE (clone UCHL1), CD154-PE (clone TRAP1), CCR7-APC , CXCR5 PerCP-Cy5.5 , and FOXP3-APC . Antibodies against surface CD3  and TCRαβ-FITC (clone WT131) were used to evaluate the expression of the CD3-TCR complex in lymphocytes. For 0.5 mg/mL polyclonal antibodies to the CD3 γ chain , the test conditions were 1 μg polyclonal antibodies in 50 μl reaction volume. Simultaneously, 3 mg/mL rabbit IgG  was used as the isotype polyclonal control, maintaining a final concentration of 1 μg IgG in 50 μl reaction volume.Peripheral blood mononuclear cells (PBMCs) were processed using Ficoll  to form a single cell suspension, which was then stained with the following monoclonal antibodies against cell surface and intracellular antigens of memory T, Treg, T follicular helper, memory B, and CD21-low B cells: anti-CD4-PE (clone SK3), CD4 FITC (clone SK3), CD8-PE (clone SK1), CD19-PerCP-Cy5.5 (clone HIB19), CD127-PE (clone hIL-7R-M21), CD25-FITC (clone 2A3), CD21-FITC , or 100 ng/mL anti-CD3 , either alone or in combination with 100 ng/mL anti-CD28  for 4 days, as described previously  and CD4+CD25low CD127hi T effector (Teff) cells. In each experiment, Teff cells were stained with 5 mM/mL carboxyfluorescein succinimidyl ester (CFSE)  for 10 min at 37°C in phosphate-buffered saline. Treg and Teff cells were then mixed at the indicated ratios based on a conventional CD4+ (Tconv) count of 5,000 cells per well, then stimulated with anti-CD2/CD3/CD28 beads  at a 1:1 Tconv:bead ratio in complete RPMI media for 4 days. In this assay, T-cell proliferation was indicated by the dilution of CFSE.To further analyze Treg cell suppression, PBMCs were fixed and permeabilized using a buffer  and then stained with monoclonal antibodies against CTLA-4  after CD4 and FOXP3 staining.RAG1, RAG2, DCLRE1C, PRKDC, and LIG4, were all wild-type (CD3G gene were based on human genome sequences (NM_000073.2) and were designed to cover the whole coding region with one pair: . The genomic DNA in exon 3  was amplified at the same time and was confirmed based on NT_033899.8 by Sanger sequencing, as previously described , azithromycin, and cefuroxime. Combined isoniazid, rifampin, and pyrazinamide were administered for 1-year for the treatment of a suspected pulmonary mycobacterial infection. The patient was not admitted for pneumonia despite exacerbations during the winter. Preseptal staphylococcus aureus cellulitis and E. coli epididymo-orchitis occurred when he was 27-years-old, and an engorged portal vein, splenomegaly, splenic artery, and coarse liver surface gradually developed , T follicular helper , and lowCD21 B cells  but a miCD3G mutation” and “immunodeficiency” revealed reports of seven patients of Turkish descent and two of Spanish descent from five unrelated families (three with consanguinity) (A PubMed search using the keywords “guinity) –12. Studguinity) , the splDel213A had neither autoimmune disorders nor any detectable autoantibodies. Patients with opportunistic infections, including parainfluenza pneumonia, Candida albicans, Giardia intestinalis, and severe EBV infection, reflect profoundly impaired lymphocyte proliferation and become candidates for transplantation , and IBD-like diarrhea had significantly higher mortality rates compared with those without  of all 10 published cases received HSCT due to refractory IBD, AIHA, and granulomatous lymphocytic interstitial lung disease or severe life-threatening infections. One of these two transplant patients, who had a p.K69X mutation, died of post-transplant respiratory failure , CD3ε (CD3E), and CD3ζ (CD247) cause the T-B+NK+ SCID phenotype, resulting in profound lymphocyte impairment and increased susceptibility to opportunistic infections, in need of rescue by HSCT. The symptoms are less severe in patients with  failure , and the failure . Four ofDel213A mutation , Freiburg Ib classification , and EuroClass group smB-CD21low (0.3% switched memory cells ≤2% and 15.5% CD21lowB cells >10%)  and the development of memory B and plasma cells. High-throughput sequencing for the TCR repertoire in Treg cells would further verify the phenotype in the absence of autoimmune disorders at a genetic and molecular level.There were some limitations to the current study. First, only 10 patients with CD3G gene deletion and presented with predominant B-cell deficiency and NRH, which mimicked the CVID phenotype but without the occurrence of autoimmune disorders; this was consistent with his normal number of Treg cells and his normal suppression function. Overall, the patients identified in the PubMed search without IBD-like diarrhea or opportunistic infections and those with autoimmune thyroiditis had better survival and were all alive at the end of follow-up. Only two patients who had severe IBD and recurrent opportunistic infections received HSCT, but they succumbed to complications.In conclusion, our patient had a novel All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by The patient and the healthy controls had provided written consent for publication of this study. All human samples were obtained under protocols approved by the Institutional Review Boards at Chang Gung Memorial Hospital (protocol 201601893A3 and 104-9578A3) and met the Institutional Review Board standards for ethical conduct in accordance with the Declaration of Helsiniki. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.W-IL and J-LH carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. C-HL and M-LK performed Treg functions. W-IL and W-LF conducted the sequence alignment. W-IL, C-HL, and J-LH designed the study and performed statistical analysis. S-HC, S-JL, L-CC, T-CY, W-ST, and T-HJ took care of the patient's critical condition. W-IL conceived the study and coordinated the investigation. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, these immunotherapeutic approaches only benefit a small fraction of cancer patients as various immune resistance mechanisms and limitations make their effective use a challenge in the majority of cancer patients. For example, adaptive resistance to therapeutic PD-1 blockade is associated with an upregulation of some additional immune checkpoint receptors. The efficacy of transferred tumor-specific T cells under the current clinical ACT protocol is often limited by their inefficient engraftment, poor persistence, and weak capability to attack tumor cells. Recent studies demonstrate that the complement receptor C3aR and C5aR function as a new class of immune checkpoint receptors. Complement signaling through C3aR and C5aR expressed on effector T lymphocytes prevent the production of the cytokine interleukin-10 (IL-10). Removing C3aR/C5aR-mediated transcriptional suppression of IL-10 expression results in endogenous IL-10 production by antitumor effector T cells, which drives T cell expansion and enhances T cell-mediated antitumor immunity. Importantly, preclinical, and clinical data suggest that a signaling axis consisting of complement/C3aR/C5aR/IL-10 critically regulates T cell mediated antitumor immunity and manipulation of the pathway ex vivo and in vivo is an effective strategy for cancer immunotherapy. Furthermore, a combination of treatment strategies targeting the complement/C3aR/C5aR/IL-10 pathway with other treatment modalities may improve cancer therapeutic efficacy.Cancer immunotherapy has made remarkable clinical advances in recent years. Antibodies targeting the immune checkpoint receptors PD-1 and CTLA-4 and adoptive cell therapy (ACT) based on  Her+ and CD8+ TILs are enhanced in these mice but also depletion of T cells through TCRα genetic deletion or antibodies against CD4+ or CD8+ T cells diminishes the enhanced antitumor immunity in the complement signaling deficient models , referred to as C3aR and C5aR1. The models for anaphylatoxins binding to their cognitive receptors have been proposed after the molecular cloning of C3aR and C5aR1 . In the on model . In maston model . In humaon model , 28. C3aon model . Similaron model –33. C5a on model –37. In aon model .+ T lymphocytes . First, it was shown that a fraction of CD8+ effectors expresses IL-10 at the peak of coronavirus infection and the IL-10+CD8+ T cells show superior CTL activity and in vivo protection against chronic infection (+CD8+ T cells (−/−Il10 reporter (Tiger) mice, CD8+ TILs within B16 tumors but not peripheral blood readily express IL-10  in patients with metastatic pancreatic cancer is being conducted based on promising efficacy data from early clinical studies. Mechanistically, checkpoint inhibition, and IL-10 treatment together enhances the number and quality of pre-existing TILs. The efficacy of PD-1/PD-L1 inhibitors is highly associated with tumor microenvironment such as TIL density, PD-1/PD-L1 expression; tumor intrinsic feature, such as tumor mutational burden, microsatellite instability; as well as gut microbiota (Although IL-10 is often associated with an immune suppressive function, recent clinical studies have unequivocally shown that IL-10 is an immune activating cytokine promoting antitumor immunity –61. In acrobiota . The cliin vitro expansion protocols of T cells for ACT. IL-2 is the primary cytokine used in the in vitro expansion of TILs and gene-engineered T cells for clinical use, however, the in vivo efficacy of expanded T cells under the current clinical ACT protocol is often limited by their inefficient engraftment, poor persistence, and weak capability to attack tumor cells (+ T cells (+ TIL proliferation (in vitro culture of TILs from human lung cancer with IL-2 drastically enhances the quantity and quality of the expanded human TILs and upregulates genes related to several signaling pathways, such TCR signaling, Notch signaling, cell cycle and CTL killing (in vitro T cell expansion protocol may improve the clinical efficacy of adoptive T cell therapy. In addition to IL-10, antagonists to C3aR/C5aR1 may also be used in such protocol as the in vitro culture of activated CD8+ T cells in the presence of C3aR/C5aR1 antagonists induces IL-10 production (Another potential application for IL-10 and C3aR/C5aR antagonists is to incorporate them into or cells –68. It w T cells –72. Consferation , 18, 61, killing . Further killing . These roduction .Significant progress has been made on testing the synergistic effect of combined treatment targeting the complement/C3aR/C5aR/IL-10 pathway and other cancer treatment modalities. The first combined strategy is dual blockade of complement signaling and immune checkpoint receptor PD-1 . The com+ T lymphocytes (+ TILs and the expansion of specific T cell clones were associated with enhanced efficacy (The second combined treatment strategy uses complement signaling blockade and chemotherapy . In a sqphocytes . Increasefficacy .+PD-1+ CD8+ T cells in the cancer patients. These exciting clinical trial results have opened new avenues for effective cancer immunotherapy.The third combination is the clinical use of pegylated rhIL-10 with the anti-PD-1 antibody pembrolizumab in a cohort of heavily pretreated patients with melanoma, no-squamous cell lung cancer or renal cell carcinoma  Figure . This co+ T cells express complement receptors C3aR and C5aR and complement signaling inhibits anti-tumor functions through repression of endogenous IL-10 production in CD8+ TILs. We and other groups have also confirmed that endogenous and exogenous IL-10 enhances anti-tumor functions of CD8+ T cells in human and mouse in vitro and in vivo. The independence of complement/C3aR/C5aR/IL-10 from the PD-1/PD-L1 signaling pathway makes it possible to block complement receptors and PD-1/PD-L1 as a combined therapy to treat cancer patients clinically. Results from other groups also suggest that the combined blockade of complement and PD-1/PD-L1 signaling with antibodies improves the efficacy of treatment through other mechanisms. Together, we and other groups provide clear evidences that complement receptors C3aR and C5aR are a new class of immune checkpoint receptors.In summary, we have identified that tumor infiltrating CD8YW, HZ, and Y-WH co-wrote the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus , rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA . For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors. In response to viral infections, host cells have evolved a plethora of so-called restriction factors that inhibit specific steps of the viral replication cycle, thereby limiting the damage caused by infections. Several of these restriction factors are encoded by interferon (IFN)-stimulated genes (ISG) , 2. The One irreversible mechanism by which viral accessory proteins counteract restriction factors is the induction of their proteolytic degradation. To achieve this, viruses frequently exploit cellular pathways of protein degradation such as the proteasome. Although several pathways may target proteins for proteasomal degradation, ubiquitination is considered the most important one . UbiquitCytomegaloviruses inhibit IFN-induced Jak-STAT signaling –20. CytoWe applied immunoprecipitation (IP) coupled to mass spectrometry (MS) to establish a comprehensive and quantitative understanding of the complexes assembled by viral DDB1/CRL-interacting proteins and their targets.Human embryonic kidney (HEK) 293T (ATCC CRL-11268) and mouse NIH3T3 (ATCC CRL-1658) cells obtained from the American Type Culture Collection were used for transfection and infection experiments, respectively. Primary mouse fibroblasts [mouse embryonic fibroblasts [MEF] and mouse newborn cells [MNC]] were isolated from C57BL/6 and BALB/c embryos and newborn mice using protocols described in Le-Trilling and Trilling . These aMurid herpesvirus 8 (“RCMV England”) were kindly provided by Sebastian Voigt, Robert-Koch-Institute Berlin and Charité Berlin. The E27 CDS fused to a C-terminal HA epitope tag was subcloned into pIRES2-EGFP by use of the NheI and EcoRI restriction sites. The CDS of pR27 of the Murid herpesvirus 2 (“RCMV Maastricht”) with a C-terminal HA epitope tag was ordered from GeneArt gene synthesis and subcloned into pIRES2-EGFP by NheI and HindIII sites. UL27 of HCMV was cloned by PCR amplification of the CDS using the primers KL-UL27-1 CGGCTAGCATGAACCCCGTGGATCAGCCG and KL-UL27-HA-2 CGGAATTCTCAAGCGTAATCTGGAACATCGTATGGGTATGTGGCGTGACCTCCGACCTC containing restriction sites and a C-terminal HA epitope tag. PCR products were cleaved with NheI and EcoRI and cloned into pIRES-EGFP2. Templates for the PCR amplification of HBx of HBV and the homologous x proteins (termed WHx7 and WHx8) of WHV were kindly provided by Mengji Lu, University of Duisburg-Essen. For the cloning into the expression vectors, primers containing restriction sites and a C-terminal HA epitope tag were used: JR-HBx-1 CGGCTAGCATGGCTGCTAGGCTGTGCTG and JR-HBx-HA-2 CGGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTAGGCAGAGGTGAAAAAGTTGCATG for HBx and JR-WHx-1 CGGCTAGCATGGCTGCTCGCCTGTGTTG and JR-WHx-HA-2: CGGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTACAGAAGTCGCATGCATTTATGCC for WHx7 and WHx8. The following primers were used for the cloning of the N-terminal HA tagged HBx: HA-HBx-1 CGGCTAGCATGTACCCATACGATGTTCCAGATTACGCTGCTGCTAGGCTGTGCTGCC and HA-HBx-2 CGGAATTCTTAGGCAGAGGTGAAAAAGTTGCATG. PCR products were cleaved with NheI and EcoRI and cloned into pIRES-EGFP2_Intron in the case of HBx and pIRES-EGFP2 in the case of WHx7 and WHx8. pUL42-HA was expressed from pcDNA3.1 (Invitrogen) and was cloned using the following primers: UL42-HA for ATCGTCAAGCTTATGGAGCCCACGCCGATGCTC and UL42-HA rev GACGATGAATTCTTACGCGTAATCTGGAACATCGTATGGGTACCCCGATGATGCTTGCGT. For the cloning of pMZ3F-STAT2-SPA, the primers STAT2-SPA for CCTCGAGA TGGCGCAGTGGGAAATGCTGC and STAT2-SPA rev GGCGGCCGCGAAGTCAGAAGGCATCAAGGGTC were used to generate a PCR product which was cleaved with XhoI and NotI and inserted into the pMZ3F vector , pIRES2-EGFP-M27-Flag [described in Trilling et al. ], pIRES2F vector , which wVpr and Vpx, both encoded by HIV-2 (TaxID 11709) Rod-9, expression plasmids were generated by Michael Emerman, Fred Hutchinson Cancer Research Center, and provided to us by Hanna-Mari Baldauf, University Hospital Frankfurt.gaussia luciferase gene.HIV-2 Rod10 vpx  and HIV-HEK293T cells were washed three times with ice-cold PBS and subsequently lysed by incubation for 1 h on ice in lysis buffer . The indProtein and IP samples were separated by classic SDS-PAGE, transferred to nitrocellulose membranes and probed with the following commercially available antibodies: rabbit α-Connexin-43 , mouse α-Itch (BD Transduction Laboratories), mouse α-HA (Sigma), rabbit α-HA (Sigma), rabbit α-DDB1 (Bethyl Laboratories Inc), rabbit α-Ncoa5 (Bethyl Laboratories Inc), rabbit α-Xpo7 (Proteintech), mouse α-Flag M2 (Sigma), or mouse α-p65 F6 (Santa Cruz Biotechnology). Xpo7 was also analyzed using an antibody which has been described previously  and whicFor in-gel digestions, 20 μl of each sample were applied to SDS-PAGE to collect the proteins in a single band for each sample. Protein bands were stained with Coomassie Brilliant Blue and subsequently digested with trypsin (SERVA) overnight at 37°C. Peptides were extracted using 50% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) and sonicated twice on ice for 10 min. Extracts were dried by vacuum centrifugation and dissolved in 34 μl 0.1% (v/v) TFA. The peptide concentration was determined by quantitative amino acid analysis performed on an ACQUITY-UPLC equipped with AccQ Tag Ultra-UPLC column (Waters). For calibration, Pierce Amino Acid Standard (Thermo Scientific) was used.Of each sample, 350 ng tryptic peptides were analyzed by LC-ESI-MS/MS on an Orbitrap Elite mass spectrometer online-coupled to an Ultimate 3000 RSLCnano system (both Thermo Scientific). After injection, peptides were trapped on a pre-column  at a flow rate of 30 μL/min (0.1% trifluoroacetic acid). After 7 min, peptides were transferred to the analytical column . A 98 min gradient of 5 to 40% of buffer B  in buffer A (0.1% formic acid) was applied to elute the peptides from the analytical column . Full MS spectra were acquired in the Orbitrap analyzer at a mass resolution of 60,000 (mass range 350–2000 m/z). Fragment mass spectra were acquired in data-dependent mode and recorded in the linear ion trap. The twenty most abundant precursor ions were selected for fragmentation using collision-induced dissociation (CID) with a normalized collision energy of 35% and an isolation width of 2.0 m/z.Protein identification was conducted with Proteome Discoverer software . Based on the human proteome file UP000005640 downloaded from Uniprot-KB (release 2015_05), a database was constructed containing 68,840 human protein sequences (UniProtKB/Swiss-Prot and UniProtKB/TrEMBL entries) and sequences of eGFP, HA-tagged proteins pM27, HBx, WHx7, WHx8, pR27, pE27, pUL27, pUL42 as well as Flag-tagged proteins Vpr and Vpx. For database searches, Sequest HT algorithm was used as implemented in the respective version of Proteome Discoverer software. Mass tolerance was set to 5 ppm for precursor ions and 0.4 Da for fragment ions. One tryptic miscleavage and variable modifications of methionine (oxidation) and cysteine (propionamide) were allowed. Peptide confidence was estimated using the Target Decoy PSM Validator function implemented in Proteome Discoverer. Peptides with a false discovery rate <1% were considered for analysis. Protein grouping function based on strict maximum parsimony principle was enabled in Proteome Discoverer.http://proteomecentral.proteomexchange.org) (Proteomics data have been deposited to the ProteomeXchange Consortium (nge.org)  via the nge.org)  with thenge.org) .Gaussia luciferase construct under the control of a constitutively active pTAL promoter for normalization, and expression vectors for HIV-2 Rod10 Vpx, HIV-1 M CH106 Vpu or dominant negative mutants of IKKα or IKKβ. To activate NF-κB, an expression vector for p65/RelA or a constitutively active mutant of IKKβ was co-transfected, or cells were stimulated with TNFα for 24 h. Two days post-transfection, a dual luciferase assay was performed and the firefly luciferase signals were normalized to the corresponding Gaussia luciferase control values.HEK293T cells were co-transfected with an NF-κB firefly luciferase reporter, a t-Test was used to compare the indicated data sets.A two-tailed and heteroscedastic (assuming different variances of the compared subpopulations) Student's Murid herpesvirus (MuHV) 8 and MuHV2, respectively. We further included the DDB1-binding protein HBx of HBV and two WHV homologs derived from genotypes 7 and 8 termed WHx7 and WHx8. Vpr and Vpx encoded by HIV-2 were also included. GFP-expressing cells served as controls. The viral proteins harboring either an HA  or a Flag epitope tag (Vpr and Vpx) were expressed in transiently transfected HEK293T cells. After overnight incubation, cells were lysed and subjected to IP using HA- or Flag-specific antibodies, and protein G sepharose, as described in the Material and Methods section. In silver stained SDS-PAGE gels, pM27, pE27, and the WHx proteins interacted with a ~125 kDa protein supposed to be DDB1 (~127 kDa)  Figure . InteresSince size alone is not a proper way to identify a protein, the immuno-purified complexes were subjected to MS to identify, quantify, and compare the interactomes of these viral accessory proteins Figure . As outlIn our analyses, we included pUL42 as outgroup control. This HCMV-encoded protein does not recruit DDB1 or CRLs, but interacts with the WW domain of the NEDD4-like Ub ligase Itch via its PPxY motif . In agreAfter definition of the outgroup, the interaction network of MCMV pM27 was assessed. As expected from previous work , pM27 efThe SDS-PAGE experiments already suggested differences regarding the strength of interaction between viral accessory proteins and DDB1. Consistently, quantitative MS revealed that pM27 retrieved DDB1 ~60-fold more efficiently than pUL27 Figure . The proFor hepadnaviral HBx and WHx proteins, the interaction with DDB1 is well-described and even substantiated by co-crystallization of DDB1 with synthetic α-helical peptides corresponding to HBx or WHx . Consistp < 0.0001) positive correlation  above background. Immunoblot experiments confirmed the specific interaction of PSME3 with pUL27  was the most abundant specifically interacting protein Figure . IntriguViruses frequently dismantle cell intrinsic immunity by evolving accessory proteins that bridge antiviral factors to proteins inducing proteolysis, e.g., DDB1 and CRLs or other components of the Ub proteasome pathway. Since CRLs became amenable for pharmacologic intervention using drugs such as MLN4924 and since CRL activity is essential for replication of several human-pathogenic viruses , 72, virHere, we comprehensively compared the interactomes of nine viral accessory proteins known or suspected to interact with DDB1 and/or CRLs and of one outgroup control protein, which did not interact with CRLs but with other Ub ligases of the NEDD4 family Figure . We founHerpesviridae)   . We conf1*log10) . Our finIn addition to hepadnaviral proteins, we also included two lentiviral proteins  in our analyses. While HIV-1 Vpr-induced apoptosis occurs in a Bax-dependent manner , a direcTo enable a comparative analysis of the interactomes of the different viral proteins, one cell line was used throughout all experiments. Due to their ubiquitous availability and broad application, we selected HEK293T cells for this purpose. One might argue that viral accessory proteins will usually be expressed in other cell types  and that this experimental approach constitutes a potential confounding factor of our data set. However, HIV can efficiently replicate in HEK293 cells once CD4 expression is enforced , suggestAs a proof of principle for the relevance of the newly identified interactions, we studied the functional consequences of the interaction between HIV-2 Vpx and p65/RelA. Based on the presence of RelA in the Vpx interactome, we tested if Vpx influences NF-κB signal transduction and found that Vpx efficiently inhibited NF-κB activation. To identify the step of the signaling cascade that is targeted by Vpx, we activated NF-κB signaling using different stimuli. Vpx antagonizes NF-κB signaling stimulated by TNFα treatment, expression of constitutively active IKKβ as well as RelA overexpression. Especially the latter result strongly suggests that the interaction between Vpx and RelA is indeed functionally relevant. NF-κB signaling is crucial for the induction and regulation of innate, adaptive, and intrinsic immune responses. Consistently, numerous viruses express proteins, which modulate NF-κB signaling . In caseTaken together, our comparative study on the interactome of viral accessory proteins known or suspected to interact with DDB1 and CRLs uncovered several new aspects of the biology of these important proteins. Furthermore, our data constitute a rich resource for future studies addressing the molecular functions and mechanisms of these viral proteins.http://proteomecentral.proteomexchange.org) (The proteomic datasets generated for this study can be found in the ProteomeXchange Consortium (nge.org)  via the nge.org)  with theCL, MR, ME, JR-A, SeH, StH, and VTKL-T did experiments. DM and BS performed MS quantifications. DH and DS analyzed the effect of HIV-2 Vpx on NF-κB activation. CL, VTKL-T, and MT wrote the paper. VTKL-T and MT supervised the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We previously profiled proteome S-sulfuration in Staphylococcus aureus (S. aureus) and identified two thioredoxin-like proteins, designated TrxP and TrxQ, that were capable of reducing protein persulfides as a potential regulatory mechanism. In this study, we further characterize TrxP, TrxQ and the canonical thioredoxin, TrxA, by identifying candidate protein substrates in S. aureus cells using a mechanism-based profiling assay where we trap mixed disulfides that exist between the attacking cysteine of a FLAG-tagged Trx and a persulfidated cysteine on the candidate substrate protein in cells. Largely non-overlapping sets of four, 32 and three candidate cellular substrates were detected for TrxA, TrxP, and TrxQ, respectively, many of which were previously identified as global proteome S-sulfuration targets including for example, pyruvate kinase, PykA. Both TrxA (kcat = 0.13 s-1) and TrxP (kcat = 0.088 s-1) are capable of reducing protein persulfides on PykA, a model substrate detected as a candidate substrate of TrxP; in contrast, TrxQ shows lower activity (kcat = 0.015 s-1). This work reveals that protein S-sulfuration, central to H2S and reactive sulfur species (RSS) signaling, may impact cellular activities and appears to be regulated in S. aureus largely by TrxP under conditions of sulfide stress.Hydrogen sulfide (H H2S andbacteria . For exaactivity . In cont (GAPDH) . Given tS-sulfuration is a reversible PTM, it remains unclear whether protein persulfide reduction, i.e., depersulfidation, occurs spontaneously by reducing reagent, or enzymatically, as in phosphorylation and S-nitrosation (2S and the reduced thiol (XXC), where X is any amino acid, to reduce disulfide bonds. As shown in Figure 1B, the N-terminal, more solvent exposed, cysteine is referred to as the “attacking” cysteine, which initiates disulfide reduction via disulfide exchange, forming a mixed disulfide intermediate between the target protein and the Trx. This is followed by attack of the C-terminal “resolving” cysteine on the N-terminal Trx Cys, with the release of the dithiol reduced target protein, and oxidized Trx. The oxidized Trx is then turned over (re-reduced) by thioredoxin reductase [TrxB in Staphylococcus aureus (S. aureus)] using a similar disulfide exchange mechanism with the reduced flavin adenine dinucleotide (FADH2) used to re-reduce TrxB disulfide and reduced nicotinamide adenine dinucleotide phosphate (NADPH) used as the final electron donor to reduce oxidized FAD .Although protein rosation . In generosation  and sulfrosation , mammalied thiol . ThioredS. aureus is also capable of reducing the persulfide on lactate dehydrogenase-2 (Ldh2) . The ultimate cellular fate of this H2S is not known but may well be assimilated by low molecular weight (LMW) thiols via a number of enzymatic and non-enzymatic processes   and the 2 (Ldh2)  in vitroed GAPDH . In vivofidation , suggestrocesses  or used S. aureus is a major nosocomial human pathogen which causes skin infections, respiratory infections and extensive mortality, with oxidation-reduction (redox) homeostasis an emerging antibacterial target against a growing resistance to commonly deployed antibiotics ; this substrate is enriched by affinity capture and identified by LC-MS/MS  was mutated to serine (C32S) by site-directed mutagenesis using primers 5′-GAGGCAGGATGGAGCCCAGACTGTCGTG-3′ and 5′-CACGACAGTCTGGGCTCCATCC-TGCCTC-3′ to create pOS1_TrxPCS. Similarly, N-terminally FLAG-tagged trxQ was cloned into pOS1 using primers 5′-CCCCCCCATATGGATTATAAAGATGATGATGATAAAATGAATAATTCATTAGACATCAAA-3′ and 5′-CCCCCCGATCCC-TATTTTAAATTTTCTAACAAATCAGTC-3′. The resolving cysteine (C31) was mutated to serine (C31S) using primers 5′-CAACGTGTGGTACTTCTAAGGTTTCAGAAAG-3′ and 5′-CTTTCTGAAACCTTAGAAGTACCACACGTTG-3′ to create pOS1_TrxQCS. In addition, C61 was mutated to serine (C61S) using primers 5′-CTTTTATCCTCAGTTTTCTAAAGATATGCAAATC-3′ and 5′-GATTTGCATATCTTTAGAAAACTGAGGATAAAAG-3′ to pOS1_TrxQCSS. N-terminally FLAG-tagged trxA was cloned into pOS1 using primers 5′-CCCCCCCATATGGATTATAAAGATGATGATGATAAAGCAATCTAAAAGTAACAGATGC-3′ and 5′-CCCCCCGATCCTTATAAATGTTTATCTAAAACTTCAGCTAAG-3′. The resolving cysteine (C32) was mutated to serine (C32S) using primers 5′-CATGGTGTGGTCCATC-TAAAATGATCGCTC-3′ and 5′-GAGCGATCATTTTAGATGGACCACACCATG-3′ to create pOS1_TrxACS.C-terminally FLAG-tagged nto pOS1  using thi constructs were first transformed to S. aureus RN4220 by electroporation and colonies selected for chloramphenicol (Cm) resistance (10 μg/mL). Plasmids were extracted from Cm-resistant colonies by QIAprep spin miniprep kit following a 30 min digestion with lysostaphin at 37°C, and then transformed into the S. aureus Newman strain by electroporation  (CS plasmid was transformed into wild-type S. aureus Newman strain.These pOS1_Trxporation . The intP/ΔtrxQ) , while ti-transformed S. aureus Newman strains were grown in 10 mL TSB medium overnight. Cells were then pelleted and resuspended in a chemically defined minimal growth medium (HHWm)   supplemeeriments ,b. Cultug for 30 s, and the supernatant collected as the flow-through (FT) sample. The resin was then washed three times with 0.5 mL wash buffer , and once with 0.5 mL lysis buffer at 4°C. The FLAG-tagged Trx-substrate protein conjugates were eluted by addition of 103 μL 5 mg/mL 3x-FLAG peptide in lysis buffer for 30 min on a roller shaker at 4°C, followed by centrifugation. 10 μL of total cell lysate, FT and wash samples, and 2 μL of eluted fraction were run on an SDS-PAGE gel to evaluate the expression of the FLAG-tagged Trx and the progress of the FLAG-enrichment strategy was confirmed by Western blot using anti-FLAG antibody (described below). The remainder of the sample was digested by trypsin in presence of 2 M urea in the same buffer overnight, with the digestion quenched by addition of 1 μL 10% trifluoroacetic acid (TFA). Peptide samples were desalted by C18 ziptip using a standard protocol, dried and resuspended in 0.1% formic acid for LC-MS/MS analysis performed in the Laboratory for Biological Mass Spectrometry in Indiana University (see below). Three biological replicates were conducted for each Trx substrate profiling experiment.Cell pellets were resuspended in 1 mL lysis buffer  and processed by five freeze-thaw cycles using liquid nitrogen and a 37°C water bath. The resuspended pellet was transferred to a 2 mL lysing matrix B  tube and shaken at 6.8 m/s for 60 s, 4°C, then spun at 13,000 rpm for 15 min, 4°C. The supernatant of ≈500 μL was added to 40 μL Anti-FLAG M2 affinity gel (Sigma-Aldrich), which was prepared by pre-washing three times with 0.5 mL TBS buffer  in a 1.5 mL microcentrifuge tube at 4°C. The cell lysate was incubated with the anti-FLAG resin for 2 h on a roller shaker at 4°C, the tube centrifuged at 5000 TM West Pico Chemiluminescent Substrate (Thermo Scientific) and exposed to film.Samples electrophoresed on an SDS-PAGE gel were transferred to a polyvinylidene difluoride (PVDF) membrane pre-blocked with 1% serum in PBS-T , and subsequently probed with monoclonal anti-FLAG M2-peroxidase (HRP) antibody produced in mouse (Sigma) in PBS-T. Western blots were imaged by SuperSignalTM 100 C18 trap column  for 6 min in 0.1% formic acid and further chromatographed on an Acclaim PepMapTM RSLC C18 analytical column  using an acetonitrile-based gradient . The elution protocol was as follows: 0–1 min, 3–7% B, linear gradient; 1–19 min, 7–40% B, linear gradient; 19–21 min, 40–100% B, linear gradient; 21–21.5 min, 100% B, linear gradient; 21.5–25 min, 100–3% B, isocratic followed by re-equilibration to 3% B. Data were collected by the Xcalibur System and converted into Mascot Generic Format by Proteome Discoverer 2.0. The extracted peak list was analyzed using Protein Prospector (UCSF).Peptide digests were analyzed using an Orbitrap Fusion Lumos mass spectrometer  equipped with an Easy-nLC 100 HPLC system. Briefly, 1 μg peptide was loaded onto Acclaim PepMapS. aureus strain Newman Trx coding sequences were subcloned into a pHis-parallel vector  (mvaS) (upp) were subcloned into the pHis-parallel vector between the NcoI and HindIII restriction sites and transformed into Escherichia coli BL21 DE3 E. coli cells. These cultures were grown in LB at 37°C, induced with 0.5 mM IPTG at an OD600 of 0.7–0.9, expressed at 16°C overnight, and cell pellets collected by low-speed centrifugation. All candidate Trx target proteins were purified using a similar protocol that involved initial solubilization of the pellet in degassed buffer A , followed by sonication. The cell lysate was centrifuged and the supernatant subjected to nucleic acid precipitation with the addition of 0.15% (w/v) polyethyleneimine (PEI), followed by centrifugation at 7,000 rpm for 15 min, 4°C. Proteins were precipitated from the supernatant with the additional of 70% (w/v) ammonium sulfate for 1 h, 4°C, and the pellets resuspended in buffer A. The resuspended ammonium sulfate pellets were filtered through a 0.2 μm filter and loaded onto a pre-equilibrated Ni-NTA column in buffer A. The column was washed with buffer A containing 50 mM imidazole, followed by elution with a gradient of 0–80% buffer B . Recombinant proteins were further purified by gel filtration chromatography on a HiLoadTM 16/600 SuperdexTM column. Fractions of >90% purity (estimated by inspection of SDS-PAGE gels) were pooled and stored at -80°C with 15% glycerol until use. The integrity of all candidate Trx substrate proteins was confirmed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, with reduced thiol contents confirmed by DTNB analysis.The l vector  to creatterparts . In addiA or PK) , 3-hydro) (mvaS)  and urac2S4 was added to 10–50 μM protein, incubated overnight at ambient temperature, and excess Na2S4 removed by buffer exchange against 25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM EDTA in the glove box. The site(s) of S-sulfuration was confirmed using the LC-MS/MS workflow described previously  for 90 min anaerobically in the dark at ambient temperature. Proteins were then digested with trypsin at 37°C overnight following the addition of 200 μL 100 mM ammonium bicarbonate buffer to the samples. These peptide samples were desalted by a C18 Omix column following a standard protocol and dried in SpeedvacTM concentrator. Cysteine-containing peptides labeled by b-IAM were then enriched on a Neutravidin resin in 100 mM ammonium bicarbonate. Non-specifically adsorbed peptides were washed from the column with 2 M NaCl in 100 mM ammonium bicarbonate, followed by elution with 10 mM TCEP at 37°C, alkylation with iodoacetamide (IAM), desalting with a C18 ziptip, and analysis by LC-MS/MS as describe above.Recombinant proteins were buffer exchanged into degassed 25 mM Tris-HCl, pH 8.0, 200 mM NaCl in the glove box. A 200-fold molar excess of Naeviously . Briefly-1 mg-1) obtained by fitting the data to a linear function. The kinetic data shown here for CstARhod-SSH as a substrate was previously reported  in 50 μL total volume for 1 h at ambient temperature, and the samples analyzed as described above. For TrxQ persulfidation experiments, 20 μM TrxQ was anaerobically incubated with a 20-fold molar excess of Na2S4 in 50 μL for 1 h at ambient temperature, with the reaction capped with IAM, digested with trypsin overnight at 37°C and quenched by 1% TFA. Peptides were desalted by C18 ziptip using a standard protocol, with the site(s) of persulfidation detected by mass spectrometry as previously described . The S. aureus strain Newman encodes multiple Trx-like proteins in addition to a canonical thioredoxin, TrxA, including TrxP, TrxQ and NWMN_1637  and TrxQ-like (CGTC) proteins are often found . We note that TrxQ harbors an active site sequence that when transplanted into Sau TrxA lowers the driving force for substrate reduction . However, other Trx-like proteins with distinct active site signatures are also found . The methicillin-resistant S. aureus (MRSA) strain FPR3757 and B. anthracis only encode TrxP- and/or TrxQ-like proteins in addition to the canonical thioredoxin . However, other bacterial genomes investigated here, including Enterococcus faecalis, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, encode thioredoxin-like proteins which harbor distinct active sites, including CGDC, CSVC, CGHC, and CAPC, with some encoding TrxP- or TrxQ-like proteins, with most of unknown function .The presence of multiple Trx-like proteins appears to be a common feature of other eduction . In any trxP/ΔtrxQ strain shows a growth phenotype upon exposure to exogenous sulfide stress, and TrxP and TrxQ are detected as S-sulfurated in cells under the same conditions while TrxA is not, we proposed that TrxP and TrxQ are specifically involved in cellular protein persulfide reduction while TrxA catalyzes disulfide reduction , candidate Trx substrates are trapped by forming an intermolecular disulfide bond with the attacking cysteine of a specific FLAG-tagged Trx, which does not resolve since the resolving cysteine is mutated to a serine. Since TrxQ has an additional single cysteine beyond the active site (C61), this cysteine was also mutated to a serine to avoid potential crosslinks to this Cys. C61S TrxQ has steady state kinetic parameters in persulfide reduction that appear comparable to that of wild-type TrxQ . Cell lysate samples were collected from mid-log cultures following addition of 0.2 mM Na2S as done previously for the proteome S-sulfuration profiling experiments . These results show that FLAG-tagged Trx is constitutively expressed and detected in the cell lysate, and that FLAG-tagged Trx remains bound to the resin during wash step, and is subsequently eluted via addition of FLAG peptide . Eluted fractions from three biological replicate lysates were then subjected to bottom-up, high-resolution LC-MS/MS and taken as representative of cellular substrate profiles for each Trx. All three Trx are constitutively expressed from the same plasmid, and are therefore expected to be expressed at similar concentrations . To approximate the relative amounts of FLAG-tagged TrxA, TrxP, and TrxQ pulled down in each of experiment, the total MS signal intensity corresponding to all identified peptides from each Trx protein was calculated from all three replicate experiments . These data reveal that TrxP was pulled-down to an extent that is lower than the other Trx proteins, approximately one-half that of TrxQ, and ≈8-fold less than TrxA. Thus, the efficiency of detecting TrxP crosslinks is in fact somewhat lower than our ability to detect TrxQ and TrxA crosslinks.The utility of this experimental strategy was first confirmed by anti-FLAG Western blotting and SDS-PAGE analysis (S. aureus Newman strain background (containing endogenous TrxA) identified four substrates in total, all of which were previously identified as S-sulfurated in cells . TrxQ substrate profiles derived from a ΔtrxP/ΔtrxQ S. aureus Newman strain background identified just three candidate substrates all of which were detected as S-sulfurated in cells, including two abundant translation factors  shared in common with the TrxP and TrxA profiles, respectively. In contrast, TrxP substrate profiles derived from the same ΔtrxP/ΔtrxQ S. aureus Newman strain background revealed crosslinks to 32 different cysteines on 25 different proteins, with 29 of these cysteines unique to TrxP and 69% previously detected as S-sulfurated in cells . These 25 candidate TrxP substrate proteins include enzymes involved in central metabolism including the major glycolytic enzymes pyruvate kinase (PykA), fructose-1,6-bisphosphate aldolase . Thus, in contrast to TrxQ and TrxA, TrxP depersulfidase activity appears to possess a significant metabolic and regulatory footprint under conditions of sulfide and RSS stress. Although it is also formally possible that cysteine-containing peptides of TrxP and a crosslink with TrxP are intrinsically easier to detect or sequence by tandem MS than those peptides derived from TrxQ or TrxA, this seems unlikely given our quantitation of peptides eluted from the anti-FLAG column, in which TrxP-derived peptides were least abundant .TrxA substrate profiles obtained from a wild-type in cells  . Thioredoxins are well known to provide electrons to Tpx to reduce reactive oxygen and reactive nitrogen adducts (S-sulfurated in cells (Table 1). These proteins (1) may be present in cells but below the detection limit of the global profiling method used previously; (2) may be cell-abundant proteins that crosslink with Trx non-specifically, e.g., ribosomal proteins L28 and L14; or (3) harbor modifications other than S-sulfuration, e.g., S-thiolations, oxidative (RSOH) or nitrosative (RSNO) modifications, that may also be reduced by thioredoxins  and the thiol-dependent peroxidase or peroxiredoxin (Tpx) . For example, C111 of 3-hydroxy-3-methylglutaryl coenzyme A synthase (MvaS), which catalyzes the rate-limiting step of cholesterol synthesis (Table 1), and represents the active site cysteine. However, it proved difficult to persulfidate MvaS in vitro with polysulfide or coenzyme A persulfide, thus preventing us from validating the TrxP-dependent persulfide reduction of MvaS (data not shown). An interesting example of substrate specificity is the metabolic enzyme, pyruvate kinase (PykA), which has four cysteine residues . Only C266 was not detected in the proteome S-sulfuration profiling experiments likely due to its short tryptic peptide .Further consideration of these Trx-conjugates reveals that 60% of the identified cysteines are invariant in Firmicutes, with some in the active site of the enzyme ; however, recombinant Sau SufB when expressed in E. coli was insoluble  biogenesis (SufB). SufB likely functions as part of a Suf BCassembly   to investigate the specific activities of TrxA, TrxP, and TrxQ toward PykA protein persulfide reduction. PykA serves as an excellent model substrate since PykA was detected reproducibly only in replicate TrxP profiling experiments and three of the four Cys are sites of protein S-sulfuration in vivo . Furthermore, mammalian PykA is readily persulfidated in vitro via reaction with inorganic polysulfides . It is unknown if C266 is S-sulfurated using this assay since the peptide is too short to be detected in either the previous or this workflow  with parameters compiled in Table 2. TrxA, TrxP, and TrxQ all possess measurable activities toward PykA-SSH, with the rank order of the three Trx in terms of maximal velocity (Vmax) found to be TrxA > TrxP > TrxQ . We next compared these catalytic parameters with those obtained previously with another S-sulfurated substrate CstARhod-SSH (Table 1). The apparent turnover number, kcat, increases ≈15-fold for TrxP only . TrxQ, on the other hand, has a relatively low apparent turnover number (kcat) toward both CstARhod-SSH and PykA-SSH, neither of which are bona fide cellular substrates . These findings are consistent with the fact that PykA is an authentic in vivo substrate for TrxP while TrxQ is not , and predicated on the expectation that the activity would be higher with an authentic cellular substrate. Finally, the catalytic efficiencies (kcat/Km) of both TrxA and TrxP are also found to increase significantly when comparing PykA-SSH to CstARhod-SSH , traced to a lower Km of TrxA for CstARhod-SSH measured previously  relative to a persulfide substrate  and saturating NADPH, and fit to the Michaelis–Menten model . Contrary to the in vivo substrate profiling results, both TrxACS and TrxPCS showed crosslinks to the same three cysteine residues of PykA in vitro , suggesting that the reduced complexity environment with the purified enzyme in isolation may not be a good model to enforce intrinsic substrate selectivity or TrxP vs. TrxQ. On the other hand, the intermolecular disulfide bond formed between the attacking cysteine on each Trx and PykA in the in vitro reaction confirms this conjugate as a mechanistic intermediate during persulfide reduction, exactly analogous to the initial steps of disulfide reduction by a canonical thioredoxin .In a further effort to recapitulate the cellular Trx substrate specificity in PykA S. aureus TrxA may be an all-purpose thioredoxin that is active against a wide range of oxidative modifications of proteome cysteines consistent with the known properties of the mammalian Trx . It also possible that TrxQ has maximal activity with another, as yet uncharacterized, thioredoxin reductase. However, it is interesting to note that it is C61, not the active site Cys pair in TrxQ, that is detected as S-sulfurated in cells . However, the biological implications of this modification are not yet known.We conclude that lian Trx . TrxP, oin cells . In vitrS. aureus strain Newman using an in vivo mechanism-based profiling strategy to trap candidate cellular substrates of TrxA, TrxP, and TrxQ. Each of these three Trx profiles are largely distinct from one another and feature non-overlapping sets of proteins save for a number of highly cell-abundant proteins involved in translation, as well as the major thiol peroxidase, Tpx. We then validate these findings using persulfidated pyruvate kinase, PykA-SSH, as a model substrate, and confirm that persulfide reduction by a Trx occurs through a mixed disulfide intermediate between the formerly persulfidated thiol and the attacking cysteine of the Trx, analogous to what occurs in disulfide reduction by TrxA , but with the release of H2S . This suggests that other cellular factors, e.g., relative protein concentrations, may augment what may be intrinsically small differences in substrate specificity in vivo.PykA was identified in the Trx profiles of TrxP , and nstrated . TrxP maS. aureus TrxA . The overall electrostatic surface potentials of the dithiol sides of these three Trx structures or models reveal that TrxA and TrxP are more similar than different, except in the immediate vicinity of the active site, which presents a weakly acidic patch in TrxP relative to the basic patch in TrxA . The surface potential around C266 is distinct from the surface potentials of the three TrxP-specific sites, in that it is more acidic relative to the basic potentials that surround C8, C144, and C410 of PykA, thus possibly consistent with a role for electrostatic complementarity in driving Trx specificity . The TrxQ model, on the other hand, suggests a strongly positive electrostatic surface potential around both the dithiol active site and C61; this may enhance the rates of reaction with what are generally negatively charged RSS at C61 . Beyond favorable electrostatics, a favorable entropy change has been implicated in the ability of Trx to interact with a range of cellular protein substrates in order to reduce their disulfide bonds . And yet, C8 and C144 are S-sulfurated in cells, and all three Cys are S-sulfurated in vitro using an inorganic (Na2S4) polysulfide donor. The regulatory impact of these modifications, however, is not yet known, but it is interesting to note that these three cysteines are capable of forming a mixed disulfide with the metabolic thiol, coenzyme A, in cells under conditions of diamide-induced oxidative or disulfide stress  (Our studies of pyruvate kinase and other candidate substrates e enzyme  a presume stress . The potgure S5) . In any HP conducted all biochemical assays. YZ and JT ran mass spectrometry samples and analyzed crosslink results. HP and DG wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vimentin (VIM) is a surface receptor for enterovirus-A71, mediating the initial binding and subsequent increase in EV-A71 infectivity. The caspid protein VP1 variation, A289T, is reportedly closely associated with less severe central nervous system (CNS) infections in humans. However, it is unclear whether VIM is associated with a reduction in CNS infections of EV-A71 in the presence of A289T. We investigated whether VIM served as a receptor for EV-A71 in the presence of an A298T substitution in VP1. EV-A71-289A and EV-A71-289T were used to infect human rhabdomyosarcoma cells, control human brain microvascular endothelial cells (HBMECs), and VIM-knockout (KO) HBMECs and inoculated BALB/c mice, SV129 mice, and VIM-KO SV129 mice. Furthermore, we cloned VP1-289A-Flag and VP1-289T-Flag proteins for co-immunoprecipitation analysis. Analysis of viral function revealed that the capacity of viral attachment, replication, and protein synthesis and secretion decreased in HBMECs during an EV-A71-289A infection, the infectivity being higher than that of EV-A71-289T upon VIM-KO. Histopathological and immunohistochemical analyses of brain tissue revealed that cerebral cortical damage was more extensive in EV-A71-289A than in EV-A71-289T infections in control SV129 mice; however, no significant difference was observed upon VIM-KO. Co-immunoprecipitation analysis revealed an interaction between VP1 and VIM, which was attenuated in VP1 harboring A289T; however, this attenuation was reversed by VIM (1-58) peptide. The A289T variation of VP1 specifically decreased the virulence of EV-A71 in HBMECs, and the attenuated interaction between VP1 harboring the A289T variation and VIM essentially decreased the CNS infectivity of EV-A71 in vitro and vivo. Human enterovirus A71 (EV-A71) is one of the major pathogens that not only causes hand, foot, and mouth disease (HFMD) but also severe central nervous system brainstem encephalitis and aseptic encephalitis . These np-selectin glycoprotein ligand 1 (PSGL-1), and scavenger receptor class B member 2 (SCRAB2) [EV-A71 infections occur through viral structural proteins including VP1 . Thus fa(SCRAB2) ,14,15. A(SCRAB2) ,17. Glia(SCRAB2) ,19,20. V(SCRAB2) . HoweverPicornaviridae. The open reading frame of EV-A71 encodes four viral capsid proteins (VP1–VP4) and non-structural proteins [EV-A71 is a non-enveloped, single-stranded, positive-sense RNA virus of the family proteins . Among tproteins . VP1 varproteins . Furtherproteins , speculaThus far, the association between the A289T variant of VP1 and the mechanism underlying EV-A71 infection in the CNS is unclear. This study aimed to analyze neuron-specific protein VIM as a potential receptor for EV-A71 to infect human brain microvascular endothelial cells (HBMECs) and the interaction between the A289T VP1 variant and VIM.Anti-EV-A71 VP1 antibody was obtained from Abnova ; anti-EV-A71 3c antibody, Abconal ; anti-vimentin antibody, Santa Cruz ; anti-β-actin antibody, anti-mouse and anti-rabbit secondary antibodies and electrochemiluminescent (ECL) Horseradish Peroxidase (HRP) substrate was used for western blotting, ; normal mouse IgG, Sigma ; the Co-Immunoprecipitation (Co-IP) Kit, ; the VIM (1–58) peptide, Ontores Biotech .2.Human rhabdomyosarcoma (RD) cells, 293T cells, human brain microvascular endothelial cells (HBMECs), control HBMECs, and VIM-knockout (KO) HBMECs were cultured in double-modified Eagle’s medium (DMEM) or 1640 medium supplemented with 10% fetal bovine serum  at 37 °C and 5% COThe full-length infectious clone of EV-A71-289A (C4) was generously provided by Prof. Qin Chengfeng . An infectious clone of EV-A71-289T (C4) was constructed via site-directed mutagenesis using the Hieff Mut Site-directed Mutagenesis kit . These two infectious clones were both transfected into RD cells, and the culture supernatant was harvested upon observation of cytopathic effects (CPEs). Two types of viruses were propagated in RD cells and stored at −80 °C ,27. To dGAPDH under the following conditions: 1 cycle at 95 °C for 30 s, followed by a three-step procedure consisting of 15 s at 95 °C, 15 s at 55 °C, and 15 s at 72 °C for 40 cycles (with data collection at the end of the 72 °C step at each cycle), and melting procedure consisting of 10 s at 95 °C, 60 s at 65 °C, and 1 s at 97 °C and cooling at 37 °C for 30 s. mRNA expression levels were normalized using the 2−ΔΔCt method and the results were expressed as an expression fold-change. To detect the level of extracellular viral RNA, at several timepoints after infection, cell supernatant was collected for viral RNA extraction with a QIAamp Viral RNA Kit . Thereafter, viral RNA was transcribed to cDNA with PrimeScript RT reagent kit with gDNA eraser . Real-l-time quantitative PCR was performed using BestarTM qPCR MasterMix  with EV-A71 primers and probe. The infectious full-length infectious clone of EV-A71 was used as template standards and the level of viral RNA was absolutely quantified. In viral attachment experiments, cells were infected with viruses at different MOI for 1 h at 4 °C. Then, the total RNA was extracted with Trizol , transcribed to cDNA with the PrimeScript RT reagent kit with gDNA eraser  and quantified using the 2−ΔΔCt method.Cells were infected at different values of multiplicity of infection (MOI) for 1 h at 37 °C. To detect the level of intracellular viral RNA, at several timepoints after infection, total RNA was extracted using Trizol  in accordance with the manufacturer’s instructions. Thereafter, 1 μg of total RNA was reverse-transcribed to cDNA, using the PrimeScript RT reagent kit with gDNA eraser , and real-time quantitative PCR was performed using BestarTM qPCR MasterMix  with EV-A71 sense primer 5-′CCAGAAGAATTTTACCATGAAGTTGT-3′ and antisense primer 5′-AGGGCTCTGCTCATACTATC-3′ and probe-CAGACGGGCACTATACAGGGAG under the following conditions: 1 cycle at 95 °C for 30 s, followed by a three-step procedure consisting of 15 s at 95 °C, 15 s at 55 °C, and 20 s at 72 °C for 40 cycles (with data collection at the end of the 72 °C step at each cycle), and cooling at 37 °C for 30 s. mRNA expression levels were normalized to those of g for 30 min at 4 °C and the supernatant was harvested. Equal amounts of protein, which was quantitated with Bioepitope Bicinchoninic Acid Protein Assay Kit  was separated via Sodium Dodecyl Sulfonate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Thereafter, proteins were electroblotted onto a polyvinylidene difluoride (PVDF) membrane . The membranes were incubated in blocking buffer (3% BSA) for 2 h at room temperature and then probed with anti-EV-A71 3c antibody and kept overnight with gentle agitation at 4 °C, followed by probing with corresponding secondary antibodies for 1 h at room temperature and with ECL reagent for exposure.At several timepoints after infection, cells were scraped off and lysed on ice for 30 min in lysis buffer . Thereafter, cell lysates were centrifuged at 15,000× To construct VP1-289A-Flag and VP1-289T-Flag proteins, the VP1 gene was amplified from the infectious clone, using the VP1 sense primer 5′-CCGGAATTCGCCACCATGGGAGATAGGGTGGCAGATGT-3′ and antisense primer 5′-CGCGGATCCCCGCTACCGCCGCTTCCCTTGTCATCGTCGTCCTTGTAATC GAGAGTGGTGATTGCTGTACG-3. Thereafter, gel purification was carried out, and the gene was cloned into the corresponding restriction sites of pEGFP-N1.Before plasmid transfection, anti-VIM antibody was immobilized on the resin in accordance with the manufacturer’s instructions. At 48 h after plasmids were transfected into 293T cells, using Lipofectamine 3000 Transfection Reagent , cell lysates were harvested and pre-cleared in accordance with the manufacturer’s instructions. Thereafter, equal amounts of cell lysates were added to the immobilized antibody and incubated overnight with gentle agitation at 4 °C. The following day, the resins were washed twice and heated in 2× sample buffer. After brief centrifugation, the supernatant was applied for western blotting.8 and 107 pfu/mL of EV-A71A and EV-A71T. Control mice were inoculated with 1640 medium. Thereafter, clinical symptoms including weakness, reduced activity, tremor, hunchback, ruffled fur, and limb paralysis were monitored [All animal protocols were approved by the Southern Medical University Experimental Animal Ethics Committee . The individual ventilated cages (IVC) system was used to offer a suitable environment for mice. BABL/c mice obtained from the Experimental Animal Centre, Southern Medical University, Guangzhou, China, control SV129 mice, and VIM-KO SV129 mice (generously provided by Prof. Huang Shenghe) aged 1 week were intraperitoneally inoculated with 10onitored ,29,30. Mt-test. p values < 0.05, from a two-tailed test was considered statistically significant.Data from at least three independent experiments were presented as mean ± SEM using SPSS 19.0 statistical software. The mean optical density (MOD) of staining intensity in immunohistochemical analyses was measured using Image Pro Plus 6.0 software. The difference among treatment groups was analyzed by a Student’s p > 0.05)   A,B. Furt > 0.05) D. A viru > 0.05) C. The vip < 0.05) (p < 0.05)  A. Extrac < 0.05) B. The EV < 0.05) D. To det < 0.05) C. These To determine whether EV-A71 could directly interact with VIM and whether there were differences in the interaction with EV-A71-289A and EV-A71-289T, viral particles were preincubated with VIM peptide or 1% DMSO for 30 min at 4 °C before infection. At 1 h after infection, total RNA was extracted, and viral RNA was quantified. In control HBMECs, viral RNA levels of both strains decreased when viral particles were preincubated with VIM peptide (1–58), suggesting that EV-A71 could directly interact with VIM. Moreover, in VIM-KO HBMECs, the viral level in infection of two strains had no difference when viral particles were preincubated with VIM peptide. A. The in8 and 107 pfu/mL of EV-A71-289A and EV-A71-289T and then monitored. The onset of infection including weakness, reduced activity, tremor, hunchback, ruffled fur, and hind limb paralysis was observed 3 to 7 d after EV-A71-289A infection and 4 to 8 d after EV-A71-289T infection . These results indicated that VIM-KO could reduce the morbidity rate of EV-A71 infections and that the A289T mutation of VP1 could decrease virulence in vivo (p < 0.05), indicating that EV-A71-289A was more neurotrophic than EV-A71-289T. This indication was further determined via histopathological and immunohistochemical analyses. In BALB/c mice and SV129 mice, histopathological analysis revealed more extensive neuronal damage and lymphocyte infiltration in the cerebral cortex in EV-A71-289A-infected mice. In particular, among SV129 mice, neuronal damage and lymphocyte infiltration in the cerebral cortex was less extensive in VIM-KO SV129 mice  peptide. Analysis of the effect of VIM (1–58) peptide on viral attachment to HBMECs seemingly displayed the same tendency, indicating that the head of VIM was critical in the interaction between VP1 and VIM. As residue 289 was located in the tail of VP1, we speculated that VP1 interacted with the head of VIM through its tail, and the A289T variation attenuated this interaction.The present study extensively investigated the association between the A289T variation of VP1 in EV-A71 and CNS infection and showed that the EV-A71 VP1 variant A289T decreased CNS infectivity of the virus via attenuation of the interaction between VP1 and VIM in vitro and vivo. Combined with our previous molecular epidemiological investigation in Guangdong Province, we speculate that EV-A71 VP1 variant A289T decreases CNS infectivity of the virus via attenuation of the interaction between VP1 and VIM. Thus, further studies from a monkey model and clinical data are required to clarify the association between EV-A71 VP1 variant A289T and less severe CNS infectivity for predicting the risk of severe EV-A71 infections. Further bioinformatics analysis for predicting the site of interaction for VP1 and VIM is needed. Moreover, this study reported that the head functional region of VIM was a hotspot for polymorphisms in the study population. Therefore, future studies are required to analyze the diversity of VIM and its interaction with VP1 variants on EV-A71 causing CNS infections.


Correction to: J Biomed Scihttps://doi.org/10.1186/s12929-015-0158-7In Fig. 3, a number of images were duplicated. Specifically, in Fig. 3a we confused the SK-MES-1 control group with the P group; and in Fig. 3c we confused the A549 control group with the A549 P group. The correct images for the entire Fig. 3 have been included below. We apologise for any inconvenience this might have caused.In Fig. After the publication of this article  it came Figure 4 SLC34A2 suppressed tumor growth and lung metastasis of NSCLC in vivo. (a) The expressions of SLC34A2 in A549-P-S, A549-P and A549 cells were confirmed by Western blot. This panel is reproduced from Yang et al. [18]. (b) The concentrations of phosphorous in the supernatant of medium were measured by using a phosphorous measurement kit in A549-P-S, A549-P and A549 cells. (c) Tumor growth curve (each group contained 8 mice). (d) Tumor wet weight. Mice were killed for measurement of tumor weight at 60 days after inoculation. (e) Lungs from mice injected with A549-P-S, A549-P and A549 cells were injected intratracheally with India ink and fixed in AAF solution. (f) The numbers of lung nodules were counted under a dissecting microscope.