Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection. Orthomyxoviridae family of enveloped viruses, which contain an eight-segmented, single-stranded (ss), negative-sense RNA genome [IAV are members of the A genome . IAV canA genome ,3,4,5. TA genome ,6,7. IAVA genome , with 18A genome ,10,11. IA genome ,13. HoweA genome ,14. In aA genome ,14. FortA genome ,14. The A genome ,16, whicA genome ,17,18. AA genome , leadingThe host innate immune system restricts IAV replication . When IAThe inflammasome is a molecular complex formed by the proteins Nucleotide oligomerization domain (NOD)-like receptor family member LRR- and Pyrin domain containing-3 (NLRP3), Apoptosis-associated Speck-like containing a caspase-recruitment domain (ASC), and pro-caspase 1 . NLRP3 iMany ISGs, such as myxovirus resistance (Mx), IFN-induced transmembrane (IFITM), protein kinase R (PKR), and 2’-5’ oligoadenylate synthetase (OAS)-ribonuclease L (RNAseL), limit IAV replication ,40. In mMembers of the interferon-induced transmembrane (IFITM) proteins family are able to prevent infection before a virus can traverse the lipid bilayer of the cell. At least three human IFITM proteins  have been described to have antiviral activity by blocking virus–host cell membrane fusion following viral attachment and endocytosis ,48. CumuActivation of OAS by dsRNA produces poly(A) chains with 2′-5′ phosphodiester bonds , which bProtein kinase R (PKR) is activated by dsRNA or by the cellular protein activator of the interferon-induced PKR (PACT), resulting in autophosphorylation and phosphorylation of cellular proteins, including the α subunit of the eukaryotic initiation factor 2 (eIF2α) . This phThe IAV genome segment 8 encodes the non-structural (NS) mRNA as a continuous primary transcript  from whiThe IAV NS1 protein allows the virus to replicate efficiently by suppressing the host innate immune response by a variety of mechanisms  , includiThe different NS1 protein domains are involved in regulating the host innate immune response, and in this review we will summarize some of the most characteristic functions. It has been shown that the NS1 RBD mutations R38A and K41A inhibit the binding of NS1 protein to dsRNA , a functThe molecular mechanisms affecting NS1’s ability to counteract innate immune responses include inhibiting RIG-I activation by sequestration of this RNA helicase and its activating ligand ,89,90, oNS1 blocks the inhibitor of kappa β kinase (IKK) subunit beta (IKK-β), inhibiting the activation of the NF-κB pathway, and preventing the expression of antiviral genes ,117 FigB. Furthe+/+ mice but replicated to high titers in lungs of PKR−/− mice and were lethal, highlighting the antiviral activity of PKR [The IAV NS1 protein directly inhibits specific ISG products, such as PKR C and riby of PKR .IAV NS1 also inhibits NLRP3 inflammasome activation ,106,107 Another important target of NS1 is the phosphatidylinositol-3-kinase (PI3K) signaling pathway. NS1 activates the PI3K pathway by direct interaction with the p85β subunit , causingNS1 proteins from some human and avian IAV strains bind to CPSF30, blocking the processing of cellular mRNAs ,110,124 The IAV NS1 protein also binds to the poly(A)-binding protein II (PABPII) , inhibitThe IAV NS1 protein also enhances the translational rate of viral, but not cellular, mRNAs . Using AThe NS1 protein of the classical swine H1N1 IAV encoded 230 amino acids until the mid-1960s, when a stop codon emerged at position 220 and caused an 11 amino acid truncation at the C-terminus . This 21The H3N2 IAV circulating in humans from ∼1950 to ∼1987 encoded an NS1 protein with a 7 amino acids C-terminal extension . No significant differences in the ability to counteract IFN responses were observed between proteins encoding the NS1 230 and 237 amino acid proteins . HoweverRecently, it was found that the NS1 protein can bind cellular dsDNA, preventing the loading of the transcriptional machinery to the DNA, decreasing the expression of antiviral genes . The resAdditionally, the NS1 of the IAV WSN binds many components of the mRNA’s export machinery: nuclear RNA export Factor 1 (NXF1), p15, Ribonucleic Acid Export 1 (RAE1), and adenovirus early region 1B-associated protein 5 (E1B-AP5), which interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex, blocking their function  Figure . These dViruses encoding truncated NS1 proteins are severely impaired in replication in cultured cells, and are highly attenuated in vivo, because of defects of the truncated NS1 proteins to counteract IFN responses. Examples include a PR8 H1N1 virus encoding the first 99 or 126 amino acids of NS1 ,129,130,Infection with a PR8 H1N1 virus encoding an NS1 protein expressing only the first 125 amino acids (PR8 NS1–125) induced significantly higher amounts of IFN-β, IL-6, C-C motif Chemokine ligand 3 (CCL3), macrophage inflammatory protein 1-alpha (MIP-1α), and tumor necrosis factor (TNF) than the parental PR8 virus. Nevertheless, the PR8 NS1–125 virus was as efficient as the parental PR8 virus in inhibiting IL-1β and IL-18 release in infected macrophages . Mutant IAV encoding amino acid changes critical for NS1-mediated host shutoff showed altered pathogenesis. For example, a single amino acid change (I106M) in the NS1 protein from an avian-derived H7N9 IAV (A/Shanghai/1/2013 and A/Shanghai/2/2013 strains) restores the NS1’s ability to bind CPSF30 and the ability to block host gene expression, including IFN response genes . FurtherIn general, the NS1 proteins of the H3N2 and H2N2 IAV bind CPSF30, inhibiting general host gene expression . HoweverThe NS1 protein encoded by pH1N1 viruses at the beginning of the 2009 pandemic did not inhibit general gene expression because of a lack of interaction with CPSF30 . InteresMoreover, it has been shown that a K186E amino acid substitution in the H3N8 CIV (A/canine/NY/dog23/2009 strain) and EIV (A/equine/Ohio/1/2003 strain) IAV restores their ability to bind CPSF30 and inhibit host gene expression ,72 Tabl.A G45R amino acid change in the NS1 protein of PR8 H1N1 contributes to virulence by increasing the expression of pro-inflammatory cytokines in mice . PR8 virIt is becoming clearer that the morbidity and pathogenesis caused by IAV are consequences of the inflammatory response . TherefoIAV infections represent a severe public health and economic concern that is most effectively prevented through vaccination . HoweverIAV lacking the NS1 gene or encoding truncations in the NS1 gene are attenuated in vivo. Therefore, they represent promising LAIV candidates for the prevention of influenza infections . For exaNS gene (NS1 and NEP) or the mRNA corresponding to the individual viral NS1 or NEP, without modifying the respective splicing and packaging signals [We have explored another strategy for generating LAIV based on NS1 modifications . We have signals . The gro signals . These rRecently, a systematic approach for vaccine development that eliminates IFN-modulating functions while maintaining virus replication fitness has been described . Using tGU CAG (amino acids RQ) are read as GUC (V), not reading the C nucleotide during PA-X translation and producing a shift in the open reading frame  in the C-terminal region of PA-X . SupportThe strain-specific loss of PA-X expression can increase or decrease viral replication and virulence. For example, loss of PA-X expression in the 2009 pH1N1 A/Beijing/16/2009 strain and in the highly pathogenic avian A/Anhui/1/2005 and A/chicken/Jiangsu/K0402/2010 H5N1 strains increased viral replication, virulence, and host inflammatory response in mice ,173 and The biological significance of PA-X’s length was studied by generating human A/Beijing/16/2009 and A/California/04/2009 pH1N1, and the avian A/tree sparrow/Jiangsu/1/2008 H5N1 and A/chicken/Hebei/LC/2008 H9N2 recombinant viruses, encoding 61 (192–252) or 41 (192–232) amino acids at the C- terminal region ,170. TheIt has been described that inhibition of PA-X expression leads to a slight virus attenuation of certain IAV strains ,166. TheThe IAV NS1 and PA-X proteins have the synergistic ability to inhibit host gene expression, although using different mechanisms. However, whereas many studies have described the individual effect of IAV NS1 and PA-X on host gene expression, the interplay between these two proteins has been barely studied. We have described that recombinant LAIV pH1N1 viruses encoding NS1 and PA-X proteins that simultaneously inhibit or do not inhibit host gene expression are impaired in viral growth properties in cultured cells, correlating with virus attenuation in vivo, in comparison to recombinant pH1N1 viruses in which only one of the viral proteins (NS1 or PA-X) inhibited host gene expression  Table 3Table 3. Multiple mutations have appeared, and they are most likely associated with host adaptation ,125,126.Importantly, these results suggest that a balance in the ability of the NS1 and PA-X proteins to regulate host cellular shutoff is beneficial for IAV , confirmWe have hypothesized that modulating the ability of NS1 and/or PA-X to counteract the innate immune responses could be a suitable strategy to generate improved LAIV . TherefoSince the MDV of the IAV LAIV remains constant between seasons, and only the viral HA and NA genes are updated , it has IAV and other viruses hijack the cellular machinery for the progression of viral infection. In order to replicate in the host, IAV encode two viral proteins, NS1 and PA-X, that have developed distinct mechanisms to counteract innate immune responses and the antiviral state produced in the infected and neighboring cells ,70,160.


JL89) and A/chicken/Zhejiang/DTID-ZJU01/2013 (H7N9ZJ13) that confer eqMx1 resistance and sensitivity respectively. A substantial reduction in Mx1 resistance was observed for the two mutations G34S and H52N in H3N8JL89 NP. Thus, eqMx1 is an important dynamic force in IAV nucleoprotein evolution. We, therefore, suggest that the amino acids responsible for Mx1 resistance should be regarded as a robust indicator for the pandemic potential of lately evolving IAVs.Interferon-mediated host factors myxovirus (Mx) proteins are key features in regulating influenza A virus (IAV) infections. Viral polymerases are essential for viral replication. The Mx1 protein has been known to interact with viral nucleoprotein (NP) and PB2, resulting in the influence of polymerase activity and providing interspecies restriction. The equine influenza virus has evolved as an independent lineage to influenza viruses from other species. We estimated the differences in antiviral activities between human MxA (huMxA) and equine Mx1 (eqMx1) against a broad range of IAV strains. We found that huMxA has antiviral potential against IAV strains from non-human species, whereas eqMx1 could only inhibit the polymerase activity of non-equine species. Here, we demonstrated that NP is the main target of eqMx1. Subsequently, we found adaptive mutations in the NP of strains A/equine/Jilin/1/1989 (H3N8 Orthmyxoviridae family and are broadly associated with acute febrile respiratory disease in many animals. Wild aquatic birds are believed to be the reservoir for these viruses [Influenza A viruses (IAVs), commonly termed flu viruses, belong to the  viruses ,2. Howev viruses ,3,4.IAV possess a segmented and negative sense single-stranded RNA genome. The eight genomic segments of IAV encode at least 10 viral proteins. IAV derives its envelope from the host cell plasma membrane and contains three transmembrane proteins: Two surface glycoproteins termed as hemagglutinin (HA) and neuraminidase (NA), along with matrix protein 2 (M2). Two non-structural proteins NS1 and NS2, collectively termed as nuclear export protein (NEP) are associated with matrix protein 1 (M1) and viral ribonucleoprotein complexes (vRNPs). The eight vRNPs comprise of eight negative-strand RNA segments associated with the nucleoprotein (NP) and three RNA-dependent RNA polymerase (RdRp) subunits  . Viral vOrthomyxoviruses such as influenza [Hantaan viruses [Myxovirus resistance proteins, including MxA and MxB in humans , belong to dynamin-like GTPases family and are considered to be cell-autonomous host restriction factors of the innate immune system against many viral pathogens. Both the type-I and type-III interferons stimulate MxA and MxB expression during innate immune signaling ,8. The Mnfluenza ,10, measnfluenza , La Crosnfluenza , and Han viruses ). HuMxA  viruses .MxA is a dynamin-like large GTPase comprised of an N-terminal globular GTPase domain, a bundle signaling element (BSE), and a C-terminal helical stalk. MxA can form stable tetramers and oligomers, which assemble in a criss-cross manner via the stalk ,16. AlthMxA represents a considerable barrier against the zoonotic introduction of avian influenza viruses into the human population . InteresThe equine influenza virus underwent an independent evolution pattern that can be compared with viruses from other species . Equine 3). The collected PBMCs were washed three times with PBS, resuspended, and maintained in RPMI 1640  supplemented with 30% horse serum and 30% fetal bovine serum (FBS) . Aliquots of these isolated PBMCs were later seeded into tissue culture flasks at a density of 5 × 106 cells/cm2 and were further incubated in a humidified chamber at 37 °C with 5% CO2. At 24 hr post-incubation, the non-adherent cells which were found floating in the medium were discarded and the adherent monocytes were again incubated for the next 3 days in order to allow their differentiation into eMDMs. The cell culture atmosphere was maintained at 37 °C with 5% CO2.Human embryonic kidney 293T (HEK293T) and Madin–Darby canine kidney (MDCK) cells were maintained in Dulbecco′s Modified Eagle′s Medium (DMEM)  supplemented with 10% foetal bovine serum  and 1% antibiotics . The equine monocyte-derived macrophages (eMDMs) were prepared from equine peripheral blood mononuclear cells (PBMCs) as described previously . BrieflyJL89) and recombinant viruses were generated using a plasmid-based reverse genetics system as previously described [50 method of Reed and Muench [Wild type Influenza A/equine/Jilin/1/89  dynamin-like GTPase 1 (Mx1), transcript variant X1, gene (GenBank accession no. XM_005606071.3), and the sequence of primers can be found in In order to clone the cDNA derived from the total RNA of eMDMs, we used reverse-transcription PCR (RT-PCR) to amplify eqMx1. The eMDMs were treated with equine IFN-α1 (100 ng/μL)  for 24 hr. The primer sets were constructed following the genome sequences of ZJ13) and H1N1 human IAV A/Sichuan/01/2009 (H1N1SC09) by Dr. Hualan Chen. The equine influenza viruses H3N8XJ07 (A/equine/Xinjiang/1/2007) and H3N8JL89 were acquired from the previously preserved viral stock in our lab.The regents used in this study were kindly provided by the following personals: H1N1 human influenza virus A/WSN/1933 (WSN) by Dr. Kawoaka, and plasmids of H7N9 A/chicken/Zhejiang/DTID-ZJU01/2013  user manual (http://www.clontech.com/CN/Products/Cloning_and_Competent_Cells/Clonin_Kits/xxclt_searchResults.jsp). Briefly, the fragments of the pCAGGS/pcDNA3.1 vector and each target gene were amplified and were then fused using the In-Fusion® HD Enzyme kit . To create the N-H7N9xJL89-C plasmid, pcAGGS-H7N9-NP was used as the template to amplify the pCAGGS vector along with the N-terminal (1–200) of H7N9. This sequence was then fused with the JL89-NP C-terminal (201–480) fragment, where pEF-JL89-NP was used to amplify the inserted C-terminal (201–480) fragments. To obtain N-JL89xH7N9-C plasmids, pEF-JL89-NP was used as the template to amplify the pEF-JL89 vector backbone sequence along with the N-terminal (1–200) of JL89 and the inserted C-terminal (201–480) fragment of H7N9 was amplified from the pCAGGS-H7N9-NP sequence. The amplified vector and insert fragments were then fused together as described earlier. Single point mutations were also created by overlapping PCR in the same way. The constructed plasmids were transformed into DH5-alpha or Stable II competent cells according to the manufacturer’s protocol . Once the transformation was successful, the plasmids were extracted using PureLink HiPure plasmids DNA purification kits . All generated plasmids were confirmed by PCR and sequencing. The sequence of all the primers is available in Site-directed mutants of NP sequences were generated using overlapping PCR and identified by DNA sequencing. Mutants of pcAGGS-H7N9-NP and pEF-JL89-NP were constructed according to the online In-FusionRenilla luciferase expression plasmids  using polyjet transfection reagent  according to the manufacturers’ protocol. Cells were maintained at 37 °C. To perform the cell lysis, we employed 200 μL of 1X reporter lysis buffer  after 24 hr of transfection. To measure the luciferase activities of Renilla and Firefly, we employed the commercially available Dual-luciferase kit  using Centro XS LB 960 luminometer . The levels of polymerase protein expression were detected using western blotting, with specific mouse monoclonal antibodies for NP and anti-HA tag antibody  for eqMx1-HA and huMxA-HA proteins. In the present study, all experimentation was performed independently at least thrice. The calculated results demonstrate mean ± SEM within one experiment.In order to determine polymerase activity, we transfected the minigenome reporter (enclosing a firefly luciferase gene linked with non-coding regions of the HA gene of influenza containing human polI as the promoter and a mice terminator sequence) with NP and viral polymerase expression plasmids. We generated the chimeric mutants of the NP gene by overlapping PCR and verified it by the sequencing technique. To predict the viral polymerase activity due to mutations in the NP, a previously described protocol was followed . BrieflyJL89 and its mutants JL89-G34S and H52N; or (3) an empty control vector (4 μg each) by using PolyJet™ In Vitro DNA transfection reagent  according to the manufacturer’s instructions. At24 hr post-transfection, co-immunoprecipitation was performed as previously described [g. Post centrifugation, using Anti-FLAG M2 magnetic beads  or Anti-NP magnetic beads  the lysates were co-incubated for 2 hr at 4 °C. Using a magnetic separator, the resins were harvested and washed thrice with cold PBS. In order to elute the resin-bound materials, we added a 3X Flag peptide and incubated the samples at 4 °C for 30 min, and then the eluted samples were boiled and SDS-PAGE was performed. The samples were shifted on the nitrocellulose membrane. 5% skim milk in Tris-buffered saline (TBS) was used as a blocking buffer for 2 hr and then the membranes were incubated with specific primary antibodies-TBST (TBS with 0.05% Tween20) at room temperature for 2 hr. Washing was performed thrice and the membranes were then incubated in TBST, containing secondary antibody  at room temperature for 1 hr. Subsequently, membranes were washed three times in PBST and scanned using a LI-COR Odyssey Imaging System .Cells were incubated in a T-75 cell culture flask  until a confluency of 70% was attained. HEK293T cells were then co-transfected with p3XFLAG-tagged eqMx1 (4 μg) together with PB1, PB2 (2 μg each), PA, FF-Luc (1 μg each), and NP expression plasmids either from (1) H7N9, and its mutants H7N9-S34G and N52H; or (2) H3N8escribed . BrieflyJL89) and/or mutant viruses (JL89-H52N-NP) at a multiplicity of infection (MOI) of 0.001. Viral dilutions were made in DMEM, supplemented with 1 μg/mL of TPCK added trypsin  and 0.25% BSA . Plates were gently shaken every 15 min so that the virus remained equally distributed within the cells. Later, excess virus was removed and cells were gently washed three times with preheated PBS and fresh infection media was added. Finally, the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection and debris was removed by centrifugation at 1000× g for 5 min. The amount of virus in each supernatant was determined either by plaque assay or by determining tissue culture infectivity dose and is expressed here either as PFU per mL or TCID50/mL respectively.The cells were seeded in 6-well plates and the next day, cells were transfected with pcDNA-3.1 eqMx1. 24 hr post-transfection, an infection experiment was performed as previously described . BrieflyTotal RNA from the collected supernatants was extracted using the RNeasy plus Minikit  and were subjected to a one-step real-time quantitative PCR (RT-qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents  according to the manufacturer’s protocol. FAM-labeled probes (EIV-Tq-P) 5′-TCAGGCCCCCTCAAAGCCGA-TAMRA and specific primers targeting the M-gene of IAV, 5′-AGATGAGYCTTCTAACCGAGGTCG-3′ (EIV-Tq-forward) and 5′-TGCAAANACATCYTCAAGTCTCTG-3′ (EIV-Tq-reverse) were used for the amplification of RNA, and relative mRNA expression levels were determined using double-standard curve methods.JL89) and/or mutant viruses (JL89-H52N-NP) at an MOI of 0.001 and collected supernatants were used to infect MDCK cells in 96-well plates at various dilutions for 48 hr. Cells were fixed with chilled absolute ethanol for 30 min. The fixed cells were incubated with the anti-NP antibody (from our lab) for 2 hr at 37 °C in a humidified chamber, followed by three washes with phosphate-buffered saline (PBS), and then incubated with fluorescein isothiocyanate- (FITC) labeled goat anti-mouse IgG  for 1 hr 37 °C. Following three washes with PBS, the cells were examined under an inverted fluorescence microscope .Briefly, MDCK cells were infected with wild-type  were retrieved from the GenBank and aligned manually using Bioedit alignment tools. The evolutionary history was inferred by the maximum likelihood method based on the JTT matrix-based model.https://www.graphpad.com/scientific-software/prism/) . Employing the statistical approaches of one-way ANOVA and Dunnett’s post-test, the statistical differences were analyzed. All experiments were thrice conducted independently. The error bars present standard deviation (SD) or standard error of the mean (SEM) in each experimental group that is documented in the figure legends. The abbreviations are designated as NS not significant, p > 0.05, * 0.01 ≤ p < 0.05, ** 0.001 ≤ p <0.01, *** 0.0001 ≤ p < 0.001, and **** p < 0.0001 respectively.In order to perform the statistical analyses, we used GraphPad Prism, version 5 , eqMx1 was predicted to be structurally similar to huMxA [ZJ13 and H5N1(R)) was slightly stronger than that of huMxA (MxA (or Mx1) as one of the major interferon-stimulated genes (ISGs) is responsible for counteracting many invading viruses, especially those belonging to to huMxA   variations between the two strains  of the H7N9ZJ13 NP, behaved like the full-length H3N8JL89 NP, indicating that the eight different AAs at the C-terminal of the NP protein did not contribute to the Mx1 resistance phenotype  contained the same amino acids at these positions as that of H7N9ZJ13, suggesting that these sites were not responsible for eqMx1 restriction  and H52N altered the restriction of eqMx1, whereas the mutants G50S, K77R, M105V, and V186I remained completely unaffected by eqMx1. The effect of these point mutations was also confirmed by creating reverse mutations in the H7N9ZJ13 NP. As expected, the reverse effect was observed for the mutation at site 52 of H7N9ZJ13 NP (N52H)  B. HoweveJL89 NP and H7N9ZJ13 NP, the specific mutations were introduced into H5N1 NP and tested for its influence on eqMx1 resistance in the H5N1 polymerase reconstitution assay. H5N1 was chosen because it is another avian IAV that is blocked by eqMx1. H5N1 contains Tyrosine (Y) at position 52 (JL89 NP (H52Y) and H5N1 NP (Y52H) showed a reverse effect against eqMx1, confirming the importance of position 52. In order to determine the effect of the two identified sites 34 and 52 in H3N8JL89 in resistance against huMxA, the polymerase activity of three H3N8JL89 NP mutants G34S, H52N, and H52Y, as well as two H7N9ZJ13 NP mutants S34G and N52H, were measured. The mutants G34S, H52N, and H52Y showed less pronounced polymerase activity than the wild type H3N8JL89 NP but still could not escape the antiviral activity of huMxA  or H7N9ZJ13 and its mutants , to mimic the viral vRNPs. Twenty four hours post-transfection, total cell lysates were harvested from transfected cells, and western blotting was performed after FLAG-IP using Anti-FLAG M2 magnetic beads . Results showed that when the eqMx1 was co-expressed with either H3N8JL89-NP or H3N8JL89-G34S-NP, no interaction was identified. Expectedly, the mutant H3N8JL89-H52N-NP showed a strong interaction with eqMx1  experiment using different NP proteins and eqMx1 protein. Flag-tagged eqMx1 was co-expressed in HEK293T cells, individually or in combination with expression plasmids of PB1, PB2, PA, viral reporter RNA (FF-luc), and NP from either H3N8th eqMx1 A. In con to–N52H B. This fJL89-H52N-NP, H7N9ZJ13-NP, and H7N9ZJ13-S34G. On the other hand, in the complex where there was no interaction between eqMx1 and NP  PB2 was co-immunoprecipitated with the said variants of NP  was done C,D and tJL89 and H3N8JL89 with an H52N substitution in the NP using a reverse genetic system as previously described [JL89 had almost the same number of copies in the presence or absence of eqMx1 during replication, whereas the number of copies of mutant virus (JL89-H52N) were drastically reduced in the cells expressing eqMx1, indicating that this mutant virus strain could be blocked by eqMx1, in contrast to the wild type H3N8JL89 virus  is essential for viral replication and to allow the virus to escape the restriction effect of eqMx1.To further evaluate the importance of 52H of the NP protein in the resistance of eqMx1, we rescued viruses H3N8escribed . Viral tescribed . MDCK ce89 virus A. Additianalysis B. From tThe interferon-induced huMxA protein signifies a key interspecies barrier for a large number of zoonotic viruses, including IAVs . Equine JL89 is predicted to be evolved from an avian origin and equine IAV epizootics has a relation to IAV epidemics in humans and canines [Equine H3N8 canines . The equ canines . The H3N canines . All thiJL89 NP, G34, and H52, which could be a possible target for H3N8JL89 IAV. In our study, we showed that in the H7N9ZJ13 NP the mutation N52H enabled it to escape the eqMx1 restriction. Interestingly, the S34G substitution in the H7N9ZJ13 did not reverse the activity against eqMx1. Substituting tyrosine (Y) at position 52 with histidine (H) in H5N1 altered the polymerase activity of H5N1 drastically and eqMx1 almost lost its restriction affect towards H5N1-Y52H. A similar substitution in H3N8JL89 NP (H3N8JL89-H52Y) produced a reverse effect and eqMx1 inhibited the polymerase activity, confirming the importance of this site. We concluded that site 52 is more universal for eqMx1 as it was found to be of equal importance in both IAV strains, whereas site 34 was found to alter the polymerase activity only of H3N8JL89 against eqMx1. The sequence alignment of unique sequences of H7N9 and H3N8 predicted that position 52 was highly conserved in both strains, whereas site 34 was more prone to mutations in both strains, reiterating the importance of site 52. None of the mutations at sites 34 and 52 in either H3N8JL89 or H7N9ZJ13 had a significant effect against huMxA but showed a slight alternation of the restriction instead. These sites may be important in escaping the huMxA antiviral activity when in combination with other as yet unidentified sites.NP is known as a key module of the vRNP complex and is indispensable for virus replication. NP is the most conserved protein within IAV viral proteins and was identified as a determinant of viral sensitivity towards Mx1 proteins in previously conducted studies, for example, site mutations at 48Q, 98K, and 99K in A/swine/Belzig/2/2001 are sufficient to provide resistance to huMxA ,22. By uThe NP and PB2 have been found to have a major role in blocking the antiviral effect of murine Mx1 ,19,20. AThe interaction of Mx1 with PB2 and NP could be direct or indirect. Other cellular proteins might be involved in this interaction and could act as a bridge between Mx1 and the vRNP complex. Only a few proteins have been reported to interact with the Mx1 protein, and these interacting proteins are potential candidates as bridging factors . MultiplThe HEK293T cell line was selected as the first choice for the transfection of plasmids throughout the study except for virus infection experiments because the HEK293T cells are well used in previous studies ,38,49,50JL89 NP determined the sensitivity and resistance to eqMx1. Further extensive studies are required to demonstrate the exact molecular mechanism involved in the structure-specific interaction and evolution, which may deliver new aims for antiviral intervention.To summarize, like its other homologs, eqMx1 could be an efficient barrier against the transmission of IAVs into the equine population. The anti-influenza activities of eqMx1 were species-specific and the NP was the major target of eqMx1. The key sites on H3N8


Wogonin promoted the ubiquitination and degradation of PI3K catalytic subunit alpha (Pik3ca), the catalytic subunit of PI3K, which was upregulated by isoprenaline treatment. Wogonin also increased the expression of neural precursor cells expressing developmentally down-regulated gene 4-like (Nedd4l), the ubiquitin E3 ligase of Pik3ca. Therefore, wogonin targets Nedd4l to induce the degradation of Pik3ca, which reverses the over-activation of the PI3K/Akt pathway and consequently relieves the isoprenaline-induced myocardial hypertrophy.Many studies have focused on identifying therapeutic targets of myocardial hypertrophy for the treatment of correlative cardiac events. Wogonin is a natural flavonoid compound that displays a potent anti-hypertrophic effect. Knowledge of its pharmacological mechanisms might reveal an effective way to search for therapeutic targets. Myocardial hypertrophy was replicated by the subcutaneous implantation of an isoprenaline mini-pump in mice or isoprenaline treatment of H9C2 cells. Pathologic changes in cardiac structure were assessed by echocardiographic and histological examinations. The signaling transduction in hypertrophy-promoting pathways and the genes involved were detected by western blot and RT-qPCR. Wogonin significantly attenuated isoprenaline-induced myocardial hypertrophy  Myocardial hypertrophy is characterized by thickening of the ventricle wall in the heart, an adaptive response to, for example, mechanical and neurohumoral stimulations . At the αs/Gβγ and Gαi/Gβγ subunits  signaling pathways both promoting myocardial hypertrophy , belong to the G protein-coupled receptor superfamily . Their ssubunits . Gαs actactility . The Gαiertrophy . TherefoFigure 1) is a natural dihydroxyl flavonoid compound isolated from the roots of Scutellaria baicalensi Georg, S. amoena C. H. Wright, or S. rivularis Wall . Isoprenaline was purchased from Tokyo Chemical Industry Co., Ltd. . All-trans-retinoic acid (RA), DBcAMP, and phorbol 12, 13-dibutyrate (PDBU) were purchased from Sigma-Aldrich LLC. . MG132 was obtained from Selleck Chemical . The vectors pUSEamp(+)/myc-tagged Akt  and pUSEamp(+)-Pik3ca were kindly provided by Liangyou Rui from the University of Michigan. The vectors pcDNA3HA and pGL3-Basic were provided by Dongping Wei from Nanjing First Hospital. The empty vector pAdeno-MCMV-MCS-3Flag and pDONR223 vector carrying a human Nedd4l gene were purchased from Obio Technology Corp., Ltd.  and Public Protein/Plasmid Library , respectively. The primers 5′-TCGAGCTCAAGCTTCGAATTCATGGAGCGACCCTATACATTT-3′ and 5′-GTCATCCTTGTAGTCGGATCCATCCACCCCTTCAAATCCTT-3′ were used to subclone human Nedd4l cDNA from pDONR223-Nedd4l by PCR. The PCR products were recombined with pAdeno-MCMV-MCS-3Flag vector cut by EcoR1/BamH1 to obtain the expression vector pAdeno-MCMV-MCS-3Flag-Nedd4l. Pik3ca cDNA was subcloned from pUSEamp(+)-Pik3ca using the primers 5′-GATCCCCCGGGCTGCAGGAATTCATGGGGAGCAGCAAGAGCAAG-3′ and 5′-ATAGAATAGGGCCCCCCCTCGAGTCAGTTCAAAGCATGCTG-3′. The PCR products were recombined with pcDNA3HA vector cut by EcoR1/Xho1 to obtain the expression vector pcDNA3HA-Pik3ca.n = 29) were divided into five groups as indicated . Under inhalation anesthesia by isoflurane, an osmotic minipump  was implanted subcutaneously on the back of the neck  were obtained from American Type Collection . H9c2 cells were cultured in Dulbecco’s modified Eagle’s high-glucose medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Hyclone) in a humidified incubator in an atmosphere of 5% CO∗LVIDs3; left ventricular end-diastolic volume (LVEDV) = 7.0/(2.4 + LVIDd)∗ LVIDd3. EF = 100∗((LVEDV-LVESV)/LVEDV). LV Mass = 1.053∗((LVIDd+LVPWd+IVSd)3-LVIDd3).After the 2-week medication, mice were anesthetized with isoflurane for echocardiographic examination. The images were obtained using a Vevo 2100 system with a 45 MHz probe  to evaluate the cardiac function and chamber size. M-mode tracings were used to measure interventricular septum diameter (IVSd), left ventricular posterior wall diameter (LVPWd), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs). Ejection fraction  and left ventricular (LV) mass were calculated as left ventricular end-systolic volume (LVESV) = 7.0/(2.4 + LVIDs)Mouse hearts were fixed in 4% paraformaldehyde solution for 48 h, dehydrated in ascending grades of ethanol, and embedded in wax. Heart sections were prepared for routine haematoxylin and eosin (H&E) staining. H9c2 cells were grown on the glass coverslips for immunofluorescence staining. Cells were washed twice with PBS, fixed for 10 min in 4% paraformaldehyde solution, and permeabilized with 0.1% Triton X-100 plus 1% bovine serum albumin (BSA) for 1 h. Then the cells were incubated with alpha-smooth muscle actin (α-SMA) antibody (1:100 dilution) for 4 h at 37°C followed by staining with a 1:100 dilution of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. A model IX72 fluorescence inverted microscope  was used to observe the cells. α-SMA antibody was purchased from Abcam . Antibody against DAPI was purchased from Sigma-Aldrich .Table 1.Total RNA extracted from mouse heart tissues and H9c2 cells were reverse-transcribed as previously described . qPCR waAs previously described , extractThe -2000 to +1 promoter region of human Nedd4l was amplified by PCR with genomic DNA from HepG2 cells using primers 5′-GGTACCGAGCTCTTACGCGTAGGTAGAGAAGCACTGACTCC-3′ and 5′-CTTAGATCGCAGATCTCGAGCGGCCGGGCTTTCC-3′. The PCR products were recombined with pGL3-Basic vector cut by Mlu1/Xho1 to obtain the reporter construct pGL3–2000/+1LUC. H9c2 cells were transfected with luciferase reporter plasmids plus the internal control vector pRL-TK-Renilla for luciferase assays in 24-well plates. After an overnight culture, cells were incubated for 24 h in complete medium supplemented with wogonin (10 μM). Luciferase activity was measured using the dual-luciferase reporter assay system  and normalized to Renilla luciferase.t-test or one-way ANOVA followed by LSD–Dunn multiple comparisons. p < 0.05 was considered statistically significant.The data and statistical analyses in this study comply with the recommendations on experimental design and analysis in pharmacology . Data arTable 2 and Figure 2A). Wogonin alone had no effects on BW or HW. The detailed anatomical changes in the heart were detected by echocardiography and reflected via hypertrophic indexes including HW, HW/BW, IVSd, LVPWd, LV Mass, LVIDd, and LVIDs. EF is to evaluate the left ventricular systolic function. Isoprenaline treatment increased HW, HW/BW, IVSd, LVPWd, and LV Mass but decreased LVIDd and LVIDs. Those changes, showing typical for concentric hypertrophy, were all reversed by wogonin. EF was neither worsened by isoprenaline nor affected by wogonin. Histologic analysis of heart section slices also revealed that wogonin attenuated the enlargement in heart size after isoprenaline treatment . Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are two well-known natriuretic peptides secreted by cardiac muscle cells when hypertrophy occurs  but did reverse the abnormal increase in heart weight (HW) . Myocardial hypertrophy was replicated by isoprenaline treatment in differentiated H9c2 cells. The mRNA levels of ANP and BNP were also investigated as hypertrophic markers. Compared to the dose of 1 μM, wogonin (10 μM) more effectively reduced the cellular mRNA levels of ANP and BNP which had been enhanced by isoprenaline treatment . In cardiomyocytes, α-SMA is a major constituent of the cytoskeleton, which determines the cell shape. Thus, staining of α-SMA was performed to observe the morphology of H9c2 cells. The average area of H9c2 cells was significantly expanded by isoprenaline treatment, while wogonin relieved this effect .The H9c2 cell line was originally derived from embryonic rat ventricular tissue. These cells are widely used as an ertrophy . H9c2 ceertrophy . The incFigures 4A,B). CREB phosphorylation was mediated by two pathways, including cAMP/PKA and PI3K/Akt, during isoprenaline treatment , implying that wogonin reduces CREB phosphorylation via the PI3K/Akt pathway instead of the cAMP/PKA pathway. PKCs were activated by its selective agonist, PDBU, which increased the mRNA levels of ANP and BNP. However, wogonin did not affect PKC-induced transcription of ANP and BNP, indicating that the anti-hypertrophy effect of wogonin is not involved in the PKC pathway .Isoprenaline induces cardiomyocyte hypertrophy via multiple pathways, including PI3K/Akt, cAMP/PKA/CREB, MAPKs , and PKC . The phoreatment . WogoninFigures 5A,B). Thus, we reasoned that wogonin specifically acts on the PI3K/Akt signaling pathway. The fact that constitutively active Akt [Akt (CA)] transfected into H9c2 cells significantly enhanced the phosphorylation of FoxO1 and FoxO3a, which were not affected by wogonin, implies that the target of wogonin is located in the upstream of Akt . As class IA PI3K, one of three types of PI3Ks, plays an important role in cardiac growth and function . Consistently, α-SMA staining showed that Pik3ca overexpression amplified the size of H9c2 cells and wogonin reversed this pathological change . When catecholamine binding to β-adrenoceptors, the G protein–coupled receptor kinase-2 (GRK2) mediates the translocation of PI3K to β-adrenoceptors and then enhances the recruitment of β-arrestin and AP-2, which finally results in the internalization and downregulation of β-adrenoceptors . Wogonin reduced the protein level of Pik3ca but failed to decrease its mRNA level . We reasoned that wogonin accelerates the degradation of Pik3ca protein. The classic protein degradation process depends on the ubiquitination-proteasome system, which includes protein-ubiquitination by ubiquitin ligase and subsequent protein-recognization by proteasomes for retrogradation. Thus, we utilized the proteasome inhibitor MG132 to prevent the ubiquitinated proteins from proteasome-mediated degradation. Wogonin induced an enhancement in the ubiquitination of plenty of proteins, which should include Pik3ca, since MG132 reversed the wogonin-induced downregulation of Pik3ca . To confirm the specificity of the wogonin-mediated ubiquitination of Pik3ca, hemaglutinin (HA)-tagged Pik3ca was transfected into H9c2 cells. The cells were then treated with wogonin and MG132. The original protein amounts of HA-tagged Pik3ca were equal in each condition and immunoprecipitation was easier to operate with HA-tagged beads. As shown in Figure 6C, the precipitated Pik3ca displayed higher ubiquitination levels after wogonin treatment.Pik3ca was significantly increased at the protein and mRNA levels in H9c2 cells with isoprenaline treatment , which led to the upregulation in protein level of Pik3ca . Besides, isoprenaline also increased the mRNA level of Pik3ca . Wogonin reversed the inhibitory effect of isoprenaline on Nedd4l expression at the transcriptional and post-transcriptional levels . We expressed Flag-tagged Nedd4l in H9c2 cells by transient transfection and found that wogonin treatment did not affect the protein levels of exogenous Nedd4l . In line with the data from cells, wogonin treatment significantly reversed the downregulation by isoprenaline on protein level of Nedd4l in mouse heart .Nedd4l is the specific ubiquitin ligase of Pik3ca, belonging to the Nedd4  family . Isoprenin vivo and in vitro. It reminds us that wogonin not only shows its therapeutic value but also can function as a probe to search for new targets for myocardial hypertrophy therapy.The present study confirms the attenuation by wogonin in isoprenaline-induced myocardial hypertrophy αs subunits to activate the classical adenylate cyclase/cAMP/PKA pathway . We also replicated the hypertrophic model with isoprenaline in differentiated H9c2 cells and found that wogonin reduced the amplified size and hypertrophic mark gene expression in H9c2 cells . Thus, the antihypertrophic effect of wogonin may depend on the amelioration in the abnormal enlargement of cardiomyocytes.Sympathetic activation is a main native factor in the progression of myocardial hypertrophy, which generates catecholamine to activate β-adrenoceptors as well as its downstream signaling and thus induce hypertrophic gene expression . There a pathway  or bind  pathway . As a β- pathway . Fourtee pathway . TherefoFigure 4). Phosphorylation of CREB can be induced by Akt or PKA . Therefore, wogonin exerts its antihypertrophic effect by suppressing PI3K/Akt pathway. The expression of ANP and BNP are also regulated by PI3K/Akt pathway through their transcriptional factor, NF-AT3, since activated Akt suppresses the downstream glycogen synthase kinase 3 beta (GSK3β), the kinase of NF-AT3, and thus provokes NF-AT3 by reducing its phosphorylation level , we reason that the target of wogonin is located in the upstream of Akt.The PI3K/Akt pathway has been linked to an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking . ActivatFigure 5). Importantly, wogonin reduced the protein level of Pik3ca and reversed the subsequent effects induced by Pik3ca overexpression, which confirms that wogonin targets PI3K. Interestingly, isoprenaline not only promotes myocardial hypertrophy via β-adrenoceptor-mediated activation of PI3K/Akt pathway, but also induces the hyperfunction of PI3K/Akt pathway through upregulating Pik3ca expression . Besides the contribution to myocardial hypertrophy, PI3K also induces internalization and downregulation of β-adrenoceptors via the interaction with GRK2, which may promote the heart failure  based on their structure and substrate specificity. The class I PI3K can be further divided into two subtypes, class IA and class IB. The mammalian class IA PI3Ks are heterodimers of a 110 kDa catalytic subunit  and a regulatory subunit of 85 or 55 kDa (p85/p55), whereas the class IB is composed of a p110γ catalytic subunit and a p101 regulatory subunit . Class I failure . Thus thFigure 6C). Nedd4l is a Pik3ca specific ubiquitin E3 ligase , suggesting that isoprenaline-mediates the up-regulation of Pik3ca not only by activating its transcription  but also by reducing its protein degradation. Wogonin enhances Nedd4l expression at the transcription level , without affecting its protein stability . It is worth emphasizing that we cannot exclude the possibility that other ubiquitin E3 ligases of Pik3ca are also regulated by wogonin. Additional ubiquitin ligases that potentially act on Pik3ca are still being searched.Wogonin downregulates Pik3ca by accelerating its degradation, since wogonin promotes the ubiquitination of Pik3a (3 ligase  whose mRFigure 8, the present investigation has revealed that wogonin targets Nedd4l and elevates its expression in cardiomyocytes, which promotes the ubiquitination and degradation of its substrate, Pik3ca. Thus, wogonin suppresses the signaling transduction in the PI3K/Akt pathway mediating the expression of hypertrophic genes, and ameliorates the myocardial hypertrophy induced by isoprenaline treatment in mice. Nowadays, β-blockers are the main drugs clinically used to treat cardiac hypertrophy and heart failure that feature over-activation of β-adrenoceptors (As shown in oceptors . In the oceptors . In thatLS, MC, and WQ contributed to experiment design. WQ, DY, JZ, QH, CT, PL, XW, PY, and QL performed the experiments and analyzed the data; WQ and DY wrote the initial draft of manuscript. LS and MC reviewed the manuscript. LS and WQ obtained the funding.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 report a seasonal increase of enterovirus D68 (EV-D68) cases in France, with 54 cases detected between 19 August and 14 November 2018. Molecular typing revealed that 20 of 32 of the isolates belonged to clade D1, only sporadically detected before in France. Median age of D1-cases was 42 years, 10 developed severe respiratory signs and one had neurological complications. The 2018-D1 viruses showed a genetic divergence of 3.34 % with D1 viruses identified previously. Since August 2018, a seasonal wave of enterovirus D68 (EV-D68) cases has been reported in France. Here, we present the molecular and clinical characteristics of the EV-D68 cases with a special focus on D1-associated cases and on severe cases.A total of 61 EV-D68 infections were reported in France from 1 January–18 December 2018, as part of the enterovirus infections national surveillance that comprises of a network of 35 hospital laboratories . SystemaOf 61 EV-D68 cases, 54 (88.5%) were detected from 19 August–14 November with a peak in cases (n = 10) in week 40 15–21 October) . We carr1 OctoberAmong the 20 D1-positive cases , the majority of infections (n = 14) were diagnosed in adults, with only six cases identified in children aged 5 years or below. Ten patients presented with severe respiratory signs including severe asthma (n = 2), acute respiratory distress (n = 4) and acute cardiorespiratory decompensation (n = 4). Among these severe respiratory cases, nine had underlying co-morbidities . Two pat3; Norm: five or less WBC per mm3; 95% of lymphocytes) but extensive microbiological investigations, including enterovirus PCR testing, were negative. Neurological symptoms improved within 10 days. Brain MRI did not show rhombencephalitis. No medullar MRI was performed. One patient in their late 60s developed neurological signs shortly after influenza-like prodromal symptoms. They presented with an acute aphasia associated with upper and lower right limb weakness and facial paralysis, compatible with cranial nerve impairment. Cerebrospinal fluid analysis was compatible with aseptic meningitis (white blood cells (WBC): 34 per mmOf the 12 B3-associated cases the median age was 4.5 years (range: 0.2–65 years). The clinical spectrum was dominated by respiratory symptoms primarily occurring in children aged 5 years or below (n = 7). Eight B3-associated cases had an underlying disease and six cases developed a severe infection, including one case of rhombencephalitis in a child aged 5 years .To characterise the global dynamics of EV-D68 clade D1 strains over time, we performed a phylogenetic analysis of the full VP1 sequences generated in this study from patients from three different administrative regions of France (GenBank accession numbers: MK121710-MK121730) in comparison to all published worldwide VP1 sequences  [In France, clade B2 and B3 largely predominated in 2014 and 2016, while clade D1 viruses were detected for the first time in 2012 (one isolate) and represented only 9 of 201 (4.5%) of the EV-D68 detected in France in 2014. The 2018 D1 isolates showed a nt divergence in the VP1 gene of 3.34% compared with the D1 strains previously characterised .According to the Bayesian skyline analysis, the 2018 outbreak remains relatively modest (within the limits of sequencing data publicly available) and has not reached the levels of the 2014 or 2016 episodes . In addiSince the global emergence of EV-D68 in 2014, this virus has been associated with a wide range of infections including neurological and severe respiratory diseases . An incrWe found that EV-D68 clade D1 was the predominant circulating genotype, partially replacing the B3 genotype that was predominant in 2016 -11. We aIn addition to severe respiratory illness, neurological complications were observed in one patient with a D1 EV-D68 infection, suggesting a neurotropic potential of this genotype. EV-D68 neurotropism was recently demonstrated in a neuroblastoma-derived neuronal cell line model, indicating that only contemporary strains of EV-D68 (including US/KY/14–18953 strain belonging to the clade D1) have acquired neurovirulence over time . EV-D68 Finally, using a metagenomic approach we provide the first European full-length genomes of clade D1 viruses, which contributes to improve our knowledge regarding EV-D68 genetic diversity. Whole genome sequence analysis did not find evidence of interclade recombination events  that couThe data presented here underline the value of extensive molecular and clinical investigations to fully describe the complex spectrum of EV-D68 infections. A global real-time surveillance of EV-D68 should be maintained and emphasised to rapidly alert epidemiologists, microbiologists and clinicians.


Bacillus subtilis EF-P also alleviates ribosome pausing at sequences encoding tandem prolines and ribosomes paused within several essential genes without a corresponding growth defect in an efp mutant. The B. subtilis efp mutant is instead impaired for flagellar biosynthesis which results in the abrogation of a form of motility called swarming. We isolate swarming suppressors of efp and identify mutations in 8 genes that suppressed the efp mutant swarming defect, many of which encode conserved ribosomal proteins or ribosome-associated factors. One mutation abolished a translational pause site within the flagellar C-ring component FliY to increase flagellar number and restore swarming motility in the absence of EF-P. Our data support a model wherein EF-P-alleviation of ribosome pausing may be particularly important for macromolecular assemblies like the flagellum that require precise protein stoichiometries.Translation elongation factor P (EF-P) alleviates ribosome pausing at a subset of motifs encoding consecutive proline residues, and is required for growth in many organisms. Here we show that  Bacillus subtilis is not required for growth but is instead required for a flagellar-mediated form of motility called swarming. By mapping spontaneous suppressors, we identify 7 broadly distributed ribosome-associated factors that, when mutated, allow swarming in the absence of EF-P, the location of which may provide mechanistic insight. Moreover, we show that EF-P enhances flagellar biosynthesis by alleviating ribosome pausing within a single flagellar structural component FliY and we implicate the RNA polymerase pausing factor NusG in long operon expression. Finally, we extend ribosome profiling analysis in the absence of EF-P to gram-positive bacteria.Translation elongation factor P (EF-P) is a highly conserved protein that alleviates ribosome pausing at consecutive proline residues. Unlike most organisms, EF-P in the bacterium  Escherichia coli, the manner in which EF-P promotes growth is thought to be pleiotropic by enhancing translation of multiple proteins that contain XPPX motifs . Fl. FlfliI ponsible ,35. Morewth rate  12,39].,39.fliI efp mutant. Cells mutated for efp were initially incapable of migrating from the site of inoculation on swarming motility agar (suppressors of efp (soe) mutations were independently isolated and each suppressor resulted in a partial restoration of swarming motility. The location of each suppressor mutation in the genome was identified using a combination of SPP1-mediated transduction linkage mapping and whole genome sequencing. The mutations were organized into 6 different classes based on their chromosomal location , mutating it closer to the σA consensus sequence   , S1A Figpromoter . We concsoes fell within two adjacent co-transcribed genes that encode the conserved 23S RNA methyltransferase homolog YacO and the ribosome-associated endonuclease Rae1  into the ydiF open reading frame , whole genome sequencing showed no other mutation in the chromosome. Both rpsC mutants however, had additional unlinked mutations , encoding the flagellar C-ring component FliY within the fla/che operon , encoding the conserved transcription elongation factor NusG  is conserved in all domains of life and has been shown to alleviate ribosome pausing at a subset of sequences encoding tandem proline residues (XPPX motifs) ,10,58. Iswarming ,13,39. Wefp mutant flagellar assembly defect is consistent with FliY being a motility-related EF-P target [fliY deletion does not perfectly phenocopy mutation of efp, as FliY is necessary for flagellar C-ring assembly and all forms of flagellar motility, whereas the efp mutant has wild type basal body numbers and can swim but not swarm  has been implicated in the direct interaction with the last protein identified by efp suppressor analysis, NusG [Suppression of the achinery . The loc  or on LB plates fortified with 1.5% Bacto agar at 37°C. When appropriate, antibiotics were included at the following concentrations: 100 μg/ml ampicillin, 10 μg/ml tetracycline, 100 μg/ml spectinomycin, 5 μg/ml chloramphenicol, 5 μg/ml kanamycin, and 1 μg/ml erythromycin plus 25 μg/ml lincomycin (mls). Isopropyl β-D-thiogalactopyranoside  was added to the medium at the indicated concentration when appropriate. Strain construction and suppressor isolation details are described in the 600 0.3–1.0) concentrated to an OD600 of 10 in PBS pH 7.4  plus 0.5% India ink. LB plates fortified with 0.65% agar were dried for 10 min open-faced in a laminar flow hood and subsequently inoculated by spotting 10 uL cell resuspensions onto the center of the plate. Plates were dried an additional 10 min open-faced in a laminar flow hood and then incubated at 37°C in a humid chamber. Swarm radius was measured along the same axis every 30 minutes.For quantitative swarm assays, strains were grown to mid log phase . GFP and Alexa Fluor 488 were visualized using a C-FL HYQ FITC Filter Cube . Images were captured with a Photometrics Coolsnap HQ2 camera in black and white and subsequently false colored and superimposed using Metamorph image software.For fluorescent microscopy of flagellar filaments and hooks, 1.0 ml of broth culture was harvested at mid-log phase, resuspended in 50 μl of PBS buffer containing 5μg/ml Alexa Fluor 488 C5 maleimide (Molecular Probes), incubated for 2 min at room temperature, and washed once in 1.0 ml of PBS buffer. The suspension was pelleted, resuspended in 30 μl of PBS buffer containing 5 μg/ml FM 4–64 (Invitrogen T13320), and incubated for 2 min at room temperature. The cells were pelleted, resuspeneded in 30 μl PBS buffer, and were observed by spotting 5 μl of suspension on a microscope slide and immobilized with a poly-L-lysine-treated glass coverslip.For fluorescent microscopy of flagellar basal bodies, 1.0 ml of broth culture was harvested at mid-log phase, resuspended in 30 μl of PBS buffer containing 5 μg/ml FM 4–64, and incubated for 2 min at room temperature. The cells were pelleted, resuspeneded in 30 μl PBS buffer, and were observed by spotting 5 μl of suspension on a microscope slide and immobilized with a poly-L-lysine-treated glass coverslip.For super-resolution microscopy, the OMX 3D-SIM Super-Resolution system with a 1.42-numerical-aperture (NA) Olympus 60X oil objective was used. FM4-64 was observed using laser line 561 and emission filter 609 nm to 654 nm, and GFP  was observed using laser line 488 nm and emission filter 500 nm to 550 nm. Images were captured using PCO Edge 5.5 sCMOS cameras, processed using SoftWorx imaging software, and analyzed using Imaris software.600 0.7–1.0 and 1 mL was harvested by centrifugation at 18,000 xg. The pellet was resuspended in 1 mL Z-buffer , 200 μg lysozyme was added, and cells were lysed at 30°C for 15 min. To obtain optical density readings within the linear range, each lysate was appropriately diluted to a final volume of 500 μL in Z-buffer. The reaction was started by the addition of 100 μL start buffer , and incubated at 30°C. The reaction was stopped by the addition of 250 μL 1M Na2CO3 and the OD420 of the mixture was measured. The β-galactosidase-specific activity was calculated according to the equation (OD420 * Dilution factor * 1000) / (time * OD600). Average β-galactosidase activity and the standard deviations for all experiments can be found in Strains were grown in LB at 37°C to ODE. coli Rosetta gami II cells and grown at 37°C in Terrific broth  to mid-log phase. 1 mM IPTG was then added and the culture was grown overnight at 16°C. Cells were pelleted, resuspended in lysis buffer (50 mM Na2HPO4 and 300 mM NaCl) and lysed using an Avestin EmulsiFlex-C3 at approximately 15,000 psi. Cell debris was pelleted by centrifugation at 31,000 xg for 30 min and Ni-nitrolotriacetic acid resin (Novagen) was added to the clarified supernatant. The resin-lysate mixture was incubated at 4°C for 3 hrs. The resin was applied to a 1-cm separation column (Bio-Rad), washed twice with 10 mL lysis buffer, and once with 10 mL wash buffer  Protein was eluted with lysis buffer containing 100 mM imidazole. To cleave the His-SUMO tag from the purified protein, ubiquitin ligase/protease was added and the reaction was incubated at room temperature for 3 hrs. To remove remaining uncleaved protein or free His-SUMO from the cleavage reaction, Ni-nitrolotriacetic acid resin (Novagen) was added and incubated at 4°C for 1 h. The resin was pelleted by centrifugation and the supernatant, containing untagged FliY or FliG, was dialyzed into PBS pH 7.4 plus 10% glycerol and stored at -20°C.The expression constructs for His-SUMO-FliY (pDP288) and His-SUMO-FliG (pKB43) were introduced into One milligram of purified FliY protein was sent to Cocalico Biologicals Inc. for serial injection into a rabbit host for antibody generation. Anti-FliY serum was mixed with FliY-conjugated Affi-Gel-10 resin (Bio-Rad 1536099) and incubated overnight at 4°C. The resin was packed onto a 1-cm column (Bio-Rad) and then washed with 100 mM glycine (pH 2.5) to release the antibody and immediately neutralized with 2M Tris base. The purification of the antibody was verified by SDS-PAGE. Purified anti-FliY antibody was dialyzed into PBS–50% glycerol and stored at -20°C.600 0.3–0.4) grown at 37°C were subjected to rapid filtration and subsequently flash frozen in liquid nitrogen. Cells were lysed in 650 μL lysis buffer , 0.5 units/μL Superase-In (Invitrogen AM2696)) using a Spex 6875 freezer mill set to 10 cycles of 2 min runs at 15 cps separated by 2 min rests. Following lysis, 25 A260 units of lysate were digested with 1500 units of S7 micrococcal nuclease (Roche 10107921001) for 1 hr at room temp after which the reaction was quenched by the addition of EGTA to a final concentration of 6 mM. The digested lysate was then applied to a 10%-50% sucrose gradient and centrifuged in a Ti-40 rotor at 35,000 rpm for 2.5 hrs at 4°C. 700 μL of fractions containing 70S ribosomes were denatured in 1% SDS and extracted once with an equal volume of 75°C acid phenol, once with an equal volume of room temp acid phenol, and RNA was precipitated with isopropanol. The precipitant was resuspended in 12 μL H2O and 25 μg RNA was mixed with 2X loading dye  and resolved on a 15% polyacrylamide TBE Urea gel. After staining the gel in SYBR Gold (Sigma S11494) for 3 min, products between ~15 and 40 bp were excised using the 10 bp O’range ladder as the standard (Thermo Scientific SM1313) and subsequently gel extracted. RNA was resuspended in H2O and the 3’ ends were dephosphorylated with T4 poly-nucleotide kinase (Lucigen 30061–1) at 37°C for 1 hr. RNA was precipitated in isopropanol and ligated to 1 μL 1 μg/μL Linker 1 (IDT /5rApp/CTGTAGGCACCATCAAT/3ddC/) with T4 RNA Ligase 2, truncated (NEB M0242S) in a 50 μL reaction at 25°C for 2.5 hrs. Products were precipitated, mixed in 2X loading dye and resolved on a 15% polyacrylamide TBE Urea gel. After staining the gel in SYBR Gold for 3 min, products between 30 and 100 bp were excised using the 10 bp O’range ladder as the standard and subsequently gel extracted. Isolated RNA was then reverse transcribed using Superscript III (Invitrogen 18080044) and 2 μL 1.25 μM reverse transcription primer (IDT 5′-(Phos)-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SpC18)-CACTCA-(SpC18)-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGC CTACAG-3′) in a 20 μL reaction for 30 min at 48C. RNA was subsequently hydrolyzed by the addition of 2.2 μL 1N NaOH and incubation at 98C for 20 min. Reverse transcription products were resolved on a 10% polyacrylamide TBE urea gel, the gel was stained in SYBR Gold for 3 min, and cDNA products were gel extracted. After resuspension in H2O, cDNA products were circularized using CircLigase (Epicentre CL4111K) in a 20 μL reaction volume at 60°C for 1 hr and subsequently heat-inactivated at 80°C for 10 min. Circularized products were precipitated in isopropanol, resuspended in H2O, and used as a template for 20 μL PCR reactions using Phusion Polymerase (NEB M0530S) with forward library primer (IDT 5′-AATGATACGGCGACCACCGAGATCTACAC-3′) and Indexed reverse library primer (IDT 5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGACTGGAGTTCAGACGTGTGCTCTTCCG-3′) where NNNNNNNN represents the barcode sequence unique to each library. After 6–10 cycles, two PCR reactions per sample with no apparent duplexed products after resolution of 2 μL on an 8% polyacrylamide TBE urea gel were pooled and DNA was purified with a QIAquick kit (Qiagen 28106) and eluted in 20 μL H2O. Libraries were sequenced using the Illumina NextSeq 500 platform in a single-end flow cell at the Indiana University Center for Genomics and Bioinformatics.Ribosome profiling libraries were prepared as described previously with minor modifications . 300 mL 2O and denatured in 1% SDS. RNA was extracted once with an equal volume of hot acid phenol, once with an equal volume of room temp acid phenol, and RNA was precipitated with isopropanol. Precipitant was resuspended in H2O and 10 μg RNA was DNAse treated using 4 units of RNase free DNase I (Invitrogen AM2222) at 37°C for 30 min in a 20 μL reaction volume. RNA was precipitated in isopropanol, resuspended in H2O and libraries were prepared by the Indiana University Center for Genomics and Bioinformatics using the ScriptSeq RNA-library kit (Illumina SSV21124). Libraries were sequenced using the Illumina NextSeq 500 platform in a single-end flow cell at the Indiana University Center for Genomics and Bioinformatics.Total RNA was extracted from the same cell lysates used to create ribosome profiling libraries. Following lysis, 2.5 A260 units were diluted in 700 μL Hth nucleotide from the 3’ end according to the 3’ assignment method described previously [http://weblogo.threeplusone.com/. Clustered orthologous group assignment was performed with the DIAMOND mapping mode of eggNOG version 4.5 [NGSutils v 0.5.9 was used to remove sequencing adapters (CTGTAGGCACCATCAAT) and filter out any reads shorter than 25 bp. Fastx v 0.0.13 was subsequently used to remove the first base from each read and resulting reads were aligned to the NCIB 3610 genome (NZ_CP020102.1) using Bowtie v 1.1.2. Using the 3primeassignment.pl script , 1,750,0sion 4.5 .E. coli ribosome profiling datasets  published by Woolstenhulme et al., 2015 were downloaded from the Sequence Read Archive and analyzed as described above using the MG1655 genome (NC_000913.3) as a reference.RNA-sequencing analysis was performed using the default parameters of the RSEM (v 1.3.0) calculate expression function and the NCIB 3610 genome (NZ_CP020102.1) as a reference . The trahttp://weblogo.threeplusone.com/. The list of FliY homolog accession numbers can be found in A local database of 2554 genomes were annotated with the Pfam library using the software hmmer v 3.1b2 and an E-value threshold of 1e-10 ,79. Prot600 of 10 in lysis buffer  and incubated at 37°C for 30 min. 6X SDS sample buffer  was added, and samples were boiled for 5 min. 12 μL boiled samples containing 2.13x107 cells  were loaded onto 12% polyacrylamide gels. To generate the standard curves, the following amounts of protein were mixed with 12 uL of the corresponding deletion mutant’s lysate and the entire mixtures were loaded onto each appropriate gel: FliY– 0.184 pmol, 0.092 pmol, 0.037 pmol, 0.018 pmol, or 0.0092 pmol. FliG– 0.22 pmol, 0.144 pmol, 0.072 pmol, 0.029 pmol, or 0.014 pmol. Lysates were resolved at 150 V for 1.25 h and transferred onto nitrocellulose membranes. For blots in which FliY concentration was analyzed, a 1:2,000 dilution of affinity purified anti-FliY antisera and a 1:2,000 dilution of affinity purified anti-FliG antisera (serving as a loading control) were used as primary antibodies. For blots in which FliG concentration was analyzed, a 1:2,000 dilution of affinity purified anti-FliG antisera and a 1:80,000 dilution of crude anti-σA antisera (serving as a loading control) were used as primary antibodies. Following incubation with the primary antibodies, nitrocellulose membranes were probed with Alexa Fluor 750-conjugated goat anti-rabbit immunoglobulin G (Life technologies A21039) and blots were imaged using a FluorChem R system. Images were analyzed using Image Studio Lite version 5.2.Strains were grown to mid-log phase, concentrated to an ODTo determine the conservation of S3, S10, NusG, YeeI, YacO, and Rae1 across all domains of life, the genomes of 191 organisms identified by Ciccarelli et al. were annotated with the Pfam library using the software hmmer v 3.1b2 and an E-value threshold of 1e-5 ,79,81. TRibosome profiling and RNA-sequencing data are available at the Gene Expression Omnibus under accession number GSE126235. Data were analyzed using custom Perl scripts.S1 TextMethods describing strain construction and suppressor isolation as well as supplementary references.(PDF)Click here for additional data file.S1 File(TAR)Click here for additional data file.S1 Figsoe2 (DK3180) and soe28 (DK6533). B) soe11 (DK5523), soe15 (DK5524), soe26 (DK5525), soe9 (DK5527), soe13 (DK5528), and soe7 (DK5526). C) soe16 (DK5900), soe22 (DK5529), soe23 (DK5531), soe12 (DK5530), and soe18 (DK5901). D) efp nusG (DK5513). E) efp flgM (DK2365), and efp flgM PIPTG-sigD (DK7073). F) WT (DK1042) and fliYS164A (DK6526).Quantitative swarm expansion assays in which mid-log phase cultures were concentrated and used to inoculate swarm agar plates. Swarm expansion was monitored along the same axis every 30 min for 6.5 hrs. Each data point represents the average of three replicates and the raw values can be found in (PDF)Click here for additional data file.S2 FigBacillus subtilis is highlighted in pink. The distribution of homologs of the Soe proteins S10 (dark blue), S3 (black), NusG (red), YeeI (gold), YacO (green), Rae1 (purple), and YdiF (cyan) across the three domains of life. Numbers indicate the following clades (1) Flavobacterium-Cytophaga-Bacteroides group, (2) Chlamydiales, (3) Planctomycetes, (4) Spirochaetes, (5) Actinobacteria, (6) Deinococcus-Thermus group and (7) Cyanobacteria. (PDF)Click here for additional data file.S3 FiglacZ to PyeeIand PyeeIsoe2. Error bars indicate the standard deviation of 3 biological replicates and the raw values can be found in PyeeI-lacZ (DK7151), PyeeIsoe2 (DK7152).β-galactosidase activity reported in Miller Units (MU) of transcriptional fusions of (PDF)Click here for additional data file.S4 Figefp. Panels E, F) Average ribosome profiling pause scores of each codon within the FliN open reading frame. The box indicates the location of the VVVV motif.The data used to generate this figure are derived from GEO accession number GSE64488. Panels A, B) Average ribosome profiling pause scores of each codon within the ValS open reading frame. The position of the PPP motif is indicated by a red asterisk on the X-axis. Panels C, D) Weighted sequence logos of amino acid sequences in which the P-site codon had a pause score of 10 or greater in the ribosome profiling datasets from WT or (PDF)Click here for additional data file.S5 Figefp (DK2050).Panels A, C) Average ribosome profiling pause scores of each codon within the ValS open reading frame. The position of the PPP motif is indicated by a red asterisk on the X-axis. Panels B, D) Average pause scores for ValS codons 28–58. The box indicates the location of the PPP motif. Error bars indicate standard deviation of 3 biological replicates. The following strains were used to generate this figure: WT (DK1042), and (PDF)Click here for additional data file.S1 TableList of the accession numbers of FliY homologs used to generate (XLSX)Click here for additional data file.S2 Table(Sheet 1) Average swarm radius in mm over time for all reported swarm assays. (Sheet 2) Hook and basal body quantifications and associated cell lengths reported in (XLSX)Click here for additional data file.S3 TablePause scores of codons in all genes with greater than 0.1 read per codon. The “position” field corresponds to the nucleotide position of the first base in the codon within the NCIB 3610 genome.(XLSX)Click here for additional data file.S4 Tableefp mutant compared to wild type. Whether or not each gene is reported as essential is indicated. The “position” field corresponds to the nucleotide position of the first base in the codon within the NCIB 3610 genome.Ribosome pause sites in which a 10-fold or greater pause score was observed in the (XLSX)Click here for additional data file.S5 TableAverage pause scores of all 8,000 tripeptides centered on the P-site (sheet 1) or E-site (sheet 2) and the number of each tripeptide’s occurrences in genes with greater than 0.1 read per codon. In all cases, only the pause score of the P-site codon was used to calculate the average.(XLSX)Click here for additional data file.S6 TableQuantification of the number of occurrences (#) and corresponding percent (%) of each cluster of orthologous groups within genes containing EF-P alleviated ribosome pause sites and all genes encoded on the NCIB 3610 chromosome.(DOCX)Click here for additional data file.S7 TableList of the accession numbers identified to be homologous to S10, S3, NusG, YeeI, YacO, Rae1, and YdiF in 191 genomes across the tree of life. These data were used to generate (XLSX)Click here for additional data file.S8 TableTranscripts per kilobase million (TMP) quantifications of each gene in the NCIB 3610 genome.(XLSX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 TableAverage ribosome densities were determined by the number of reads mapping to a particular gene divided by the number of codons within that gene. Only genes in with an average ribosome density greater than 0.1 in all 12 ribosome profiling libraries were analyzed.(XLSX)Click here for additional data file.


Neuroinvasive viruses, such as alpha herpesviruses (αHV) and rabies virus (RABV), initially infect peripheral tissues, followed by invasion of the innervating axon termini. Virus particles must undergo long distance retrograde axonal transport to reach the neuron cell bodies in the peripheral or central nervous system (PNS/CNS). How virus particles hijack the axonal transport machinery and how PNS axons respond to and regulate infection are questions of significant interest. To track individual virus particles, we constructed a recombinant RABV expressing a P-mCherry fusion protein, derived from the virulent CVS-N2c strain. We studied retrograde RABV transport in the presence or absence of interferons (IFN) or protein synthesis inhibitors, both of which were reported previously to restrict axonal transport of αHV particles. Using neurons from rodent superior cervical ganglia grown in tri-chambers, we showed that axonal exposure to type I or type II IFN did not alter retrograde axonal transport of RABV. However, exposure of axons to emetine, a translation elongation inhibitor, blocked axonal RABV transport by a mechanism that was not dependent on protein synthesis inhibition. The minority of RABV particles that still moved retrograde in axons in the presence of emetine, moved with slower velocities and traveled shorter distances. Emetine’s effect was specific to RABV, as transport of cellular vesicles was unchanged. These findings extend our understanding of how neuroinvasion is regulated in axons and point toward a role for emetine as an inhibitory modulator of RABV axonal transport. Rabies virus (RABV) and alpha herpesviruses (αHV) (e.g. herpes simplex virus) evolved to enter the nervous system efficiently each time they infect a host. In most mammals, RABV reaches the brain, causing a fatal encephalitis. Whereas, αHV remain in the peripheral nervous system in a quiescent but reactivatable state. Despite distinct clinical outcomes, both RABV and αHV must invade axons and repurpose the axon transport machinery to travel long distances toward the neuronal cell bodies where virus replication occurs. How virus particles hijack the transport machinery and how axons respond to and regulate infection are questions of significant interest. We investigated how axonal RABV transport is regulated by exposing axons to interferons or protein synthesis inhibitors, both of which restrict transport of αHV particles. Unlike αHV infection, exposure of isolated axons to interferons has no effect on RABV neuroinvasion. However, RABV transport is blocked by axonal exposure to the translation elongation inhibitor, emetine, via a mechanism that does not depend on protein synthesis inhibition. The effect of emetine is not due to a global inhibition of axon transport because emetine does not limit axonal transport of cellular vesicles. Therefore, emetine may be a novel inhibitory modulator of RABV axonal transport. Rhabdoviridae family. A zoonotic rabies infection begins with the bite of an infected animal into a muscle, followed by spread of virus particles through peripheral nervous system (PNS) somatic motor neurons and into the central nervous system (CNS). Spread of the infection from the CNS to salivary glands facilitates transmission to other hosts   was used at 1:100 for IF. Rabbit polyclonal anti-RABV N (raised against purified ribonucleoprotein particles) was made by the Schnell Lab and used at 1:1000 for WB. Monoclonal anti-RABV P antibody was a gift from Danielle Blondel  and usedEmetine dihydrochloride hydrate  was used at 100 μM (dissolved in water) unless otherwise specified. Cycloheximide (CHX)  was used at 100 μg/ml (dissolved in DMSO), and Puromycin  was used at 10 μg/ml (dissolved in DMSO). Emetine, CHX, or Puromycin were added 1 h prior to infection unless otherwise specified. For the untreated/ no treatment controls, the appropriate solvent (water or DMSO) was added 1 h prior to infection unless other timing is specified. Recombinant rat IFN-gamma protein  and recombinant rat IFN-beta  were used at 500 U/ml (retrograde infection) or 1000 U/ml (particle tracking). IFN treatment was initiated 24 h prior to infection. SYTOX Green Nucleic Acid Stain  was used at 5 nM for 10 min to label dead cells. Fluorescent lipophilic dyes  were used at 1:1000 dilution to label cell bodies with axonal connection to the N compartment.N2c P: 5’-TTGGCAAAGAATTCGTCTCCGTACGACCATGAGCAAGATCTTTGTTA-3’ (fwd) and 5’-GAGCCGTCGCCGGAGCAGGATGTATAGCGATTC-3’ (rev);mCherry: 5’-TACATCCTGCTCCGGCGACGGCTCTGGCATGGTGAGCAAGGGCGAG-3’ (fwd) and 5’-GAAAACTCGGTTACTTGTACAGCTCGTCCATG-3’ (rev);N2c M-IGR: 5’-GTACAAGTAACCGAGTTTTCGAACTCAGTC-3’ (fwd) and 5’-GGGAAAAAGATCTCGTCTCGCTAGCCTTCCCGGGGTCTTTTGAG-3’ (rev)The RABV recombinant was derived from the highly neuroinvasive and neurotropic CVS-N2c parental strain . PCR priPCR products contained 15–40 bp of overlapping ends to facilitate fragment assembly. The stop codon of P was mutated, and mCherry was fused to the C-terminus with a 5 amino acid spacer sequence (GGG GAC GGC TCT GGC) inserted between the end of P and the start of mCherry. PCR products were ligated into pCAGGS vectors using NEBuilder HiFi DNA Assembly Cloning Kit  following the recommended protocol for assembly of DNA inserts into linear vectors. Briefly, the digested pCAGGS vector and the 3 PCR products were added to the HiFi DNA Assembly master mix in equimolar amounts (0.1 pmol/fragment) and incubated at 50°C for 1 h. The ligation product was transformed into NEB5α competent cells  following the manufacturer’s protocol, and minipreps were prepared from single colonies. P-mCherry-M-IGR inserts were verified by restriction digest and DNA sequencing. P-mCherry expression was confirmed by fluorescence microscopy after transient transfection of the mammalian expression vector into N2a cells.Verified inserts were digested out of the pCAGGS subcloning vector by cutting at the SpeI site (C-terminus of P) and XmaI cloning site (flanking the 5’end of the glycoprotein gene), and inserts were gel purified. The P-mCherry-M-IGR insert was ligated into the SpeI/XmaI-digested N2c RABV genomic cDNA using T4 ligase  overnight at room temperature (RT). Ligated genomes were transformed into NEB5α cells, and single colonies were picked for mini preps.For RABVΔG, N2c RABV genomic cDNA was digested with SpeI and NheI (cloning site flanking the 3’-end of the glycoprotein gene) to remove the genomic region from the C-terminus of P to the end of the G gene. P-mCherry-M-IGR was digested out of the pCAGGS expression vector and re-ligated into the SpeI/NheI-digested N2c genomic cDNA. P-mCherry insertion and G deletion were confirmed by restriction digest, and maxi preps were produced from verified clones prior to virus recovery.2 flasks for the remainder of the virus recovery period. The cells were kept at 34°C for the duration of virus recovery and passaged as needed with media change every 4 days.The RABV recombinant was recovered on a complementing cell line expressing N2c G (NG cells). The cells were made by transfecting N2a cells with plasmid vectors pTET-off  and pTRE2Hyg  containing the N2c glycoprotein gene. Clones were selected with Geneticin and Hygromycin. Resistant clones were cultured in the absence of doxycycline and screened by immunofluorescence staining with polyclonal antibody against the RABV glycoprotein. Two clones that stained positive for RABV-G were selected (NG7 and NG13) for setting up virus recovery and for large scale virus growth. Briefly, NG13 cells in 6 well dishes were transfected with 1.7 μg of full length genomic cDNA plus 2.5 μg of a mix of pTIT-N, pTIT-P, pTIT-L, and pCAGGS-T7 combined in a 4:2:1:2 ratio, respectively. Plasmids (pTIT) expressing the SAD B19 N, P, and L proteins were provided by Matthias Schnell . The DNARed fluorescent foci were visible as early as 8 days post transfection, and all cells expressed P-mCherry by approximately 21 days post transfection. Supernatants were collected for virus stocks at days 21, 24, and 28 post transfection. Supernatants were spun for 20 min at 3000 rpm (4°C) to pellet cell debris. Supernatants were filtered through 0.45 μm filters and concentrated using Amicon Ultra -15 Centrifugal Filter Units (100KDa NMWL) . Concentrator units were spun for 20 min at < 4000 x g. Concentrated virus stocks were kept at 4°C for immediate use or -80°C for long term storage. Each virus stock was titered in triplicate on N2a cells to determine the concentration (ffu/ml).Concentrated virus stocks were overlaid on a 20% sucrose cushion in either 13.2 ml  or 38 ml  thin wall, ultra-clear tubes. Tubes were spun in a Beckman Coulter ultracentrifuge at 25,000 rpm for 1.5 h using the either the SW 41 Ti (13.2 ml tubes) or SW 32 Ti (38 ml tubes) swinging bucket rotor . The supernatant was aspirated, the remaining liquid decanted, and the tubes were spun upside down in 50 ml conical tubes to remove any residual liquid. Pellets were resuspended in PBS at 4°C overnight. 6% sucrose was added for cryopreservation.Adenoviral vectors were constructed by Gateway recombination into pAd/CMV/V5-DEST vectors . GFP was fused to the N-terminus of Rab5 or Rab7 as described previously .Dissociated SCG neurons or cell bodies from the S compartments were lysed in RIPA light buffer  NP40; 0.1% (w/v) SDS; 0.1% (v/v) Triton X-100) supplemented with 1 mM DTT and protease inhibitor cocktail . Lysates were incubated on ice for 20 min and centrifuged at 10,000 rpm (4°C) for 5 min. Supernatants were mixed with 5x laemmli buffer and heated for 10 min at 90°C. Axons from N compartments were directly lysed in 2x laemmli and heated. Proteins were separated by SDS-PAGE on 4–12% NuPAGE Bis/Tris gels. Proteins were transferred to nitrocellulose membranes  using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Membranes were blocked in 5% non-fat dry milk powder in PBS-T  for 1 h at RT. Primary antibody was diluted in 1% milk powder in PBS-T. Membranes were incubated with primary antibody overnight at 4°C followed by PBS-T washes (3 x 10 min). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (KPL) were diluted in 1% milk powder in PBS-T. Secondary antibody was added to the membrane for 1 h at RT followed by three PBS-T washes. Chemiluminescent substrate, Supersignal West Pico or West Dura (Pierce), was added to the membrane for 5 min. Protein bands were visualized by exposing the blot on HyBlot CL autoradiography film . For detection of viral proteins in sucrose purified particles, proteins were separated by SDS-PAGE on 10% polyacrylamide Tris/Glycine gels. Primary anti-RABV antibodies were diluted in 10% BSA, and secondary antibodies were diluted in PBS-T + 5% milk powder.For immunofluorescence staining of P-mCherry-positive RABV particles, supernatants were collected from G-expressing N2A cells (NG cells) at 7 d post RABV P-mCherry infection (MOI 0.1). Following filtration, concentration, and purification (20% sucrose cushion in PBS), supernatants were spotted on glass coverslips and left to adsorb to the glass for 15 min at 37°C. Particles were blocked without fixation for 1 h in DMEM + 10% FBS followed by 1 h staining with 1° antibody recognizing the RABV G protein and Alexa Fluor 488-conjugated 2° antibody (green). Particles were washed 3 times with DMEM for 5 min per wash prior to and after 2° antibody staining.For immunofluorescence of dissociated SCG or M compartment axons, cells were fixed in 4% PFA at RT for 10 min, permeabilized in 0.25% Triton X-100 for 20 min at RT, and blocked in 3% BSA-PBS for 1 h. FITC-conjugated anti-RABV (N) antibody was diluted in 3% BSA-PBS and added to cells for 1 h followed by three 5 min washes in PBS + 0.05% Tween. DAPI was added to dissociated SCG in the third wash for 5 min.5 ffu of RABV P-mCherry unless otherwise specified. At 5 hpi, green lipophilic dye (DiO) was added to the N compartment to label the connected cell bodies in the S compartment. Using live cell imaging, the entire S compartment was tile imaged at designated times post axonal infection for P-mCherry expression (red) and DiO staining (green). Total numbers of green and dual-colored cell bodies were counted manually using NIS Elements Advanced Research software (Nikon). The average number of DiO-positive cell bodies counted per chamber (n = 150 chambers) was 1981 cells. The % infected cell bodies refers to the percentage of DiO-positive cell bodies that were also P-mCherry-positive.Unless otherwise specified, N compartment axons were either untreated (appropriate solvent added to control), pretreated with IFNβ or IFNγ for 24 h, or pretreated with protein synthesis inhibitors for 1 h prior to axonal infection with RABV P-mCherry see . N compaFor protein synthesis inhibitor experiments, the inhibitors and virus inoculum were washed out and removed from the N compartment at 5 hpi, at which point the N compartment was replaced with fresh neurobasal media. For the IFN experiments, IFN and virus inoculum were not removed from the N compartment, and media was not replaced throughout the duration of the experiment.5 ffu). Time lapse imaging was used to visualize and count the number of RABV particles that moved from N into M between 2–4 hpi. Each movie captured one field of view (FOV) for 15 seconds (sec) at > 10 frames/sec. For Rab5 and Rab7 particle tracking, S compartment cell bodies were transduced with adenoviruses expressing either Venus-Rab5 or Venus-Rab7. At 4 days post transduction, the N compartment axons were either untreated or treated with 100 μM emetine (in the absence of RABV infection). At 2–4 h post emetine treatment, Rab motility was recorded in N compartment axons. The percentage of moving Rab particles to total particles (moving + stationary) was calculated per axon for a minimum of 22 axons per condition.For RABV particle tracking, N compartment axons were either untreated (control), pretreated with IFNβ or IFNγ for 24 h, or pretreated with 100 μM emetine 1 h prior to RABV P-mCherry infection in N (10Imaging was conducted on a Nikon Eclipse Ti inverted epifluorescence microscope using a Photometrics CoolSNAP ES2 CCD camera or an Andor iXon3 EMCCD camera (particle tracking). Images and movies were processed using NIS Elements Advanced Research software (Nikon) and Fiji Image J . Comparawww.graphpad.com). Figure legends specify the statistical test applied for each analysis, as well as the measure of dispersion about the mean and the number of replicates used. Experiments were repeated three times unless otherwise specified. Standard deviation (SD) is reported as the error for all measurements in the results section except for particle velocities and particle track lengths. For those population level measurements, the standard error of the mean (SEM) is reported. Differences were considered statistically significant when p values were less than 0.05 .All data were analyzed with Graphpad Prism 7.04  of Princeton University Research Board. The IACUC approved all animal experiments (protocol # 1947). Animals were euthanized by carbon dioxide inhalation, as recommended by the American Veterinary Medical Association (AVMA) guidelines on euthanasia. All personnel adhered to applicable federal, state, local, and institutional laws and policies governing ethical animal research. This includes the Animal Welfare Act (AWA), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training, and the Health Research Extension Act of 1985.S1 Fig(A) PRV UL25-mCherry, RABV P-mCherry, and PRV mCherry-UL35 particles from sucrose-purified supernatants . (B) Quantification of particle intensity (arbitrary units (AU)) based on mCherry brightness across individual particles of PRV UL25-mCherry (n = 815), RABV P-mCherry (n = 825), and PRV mCherry-UL35 (n = 841). Error bars indicate mean maximum particle intensity + SD. (C) Table listing the approximate average copy number per particle of RABV P and PRV pUL35. Protein copy number was extrapolated from the known PRV pUL25 copy number (60 copies) by comparing the mCherry fluorescence emission intensities between virus strains.(TIF)Click here for additional data file.S2 FigN compartment axons were treated with IFNβ or IFNγ for 24 h. S and N compartments were lysed separately, and proteins were separated by SDS-PAGE on 4–12% gradient gels. Phosphorylated STAT1 (P-STAT1) levels were determined in the S and N compartments by western blotting. Symbols indicate the presence (+) or absence (-) of IFN in the N compartment. Β-actin served as a loading control.(TIF)Click here for additional data file.S3 Fig(A) Experimental setup for immunofluorescence (IF) staining of RABV particles in the M compartment. (B) IF staining of P-mCherry-positive particles (red) using FITC-conjugated anti-RABV antibody targeting the N protein. White arrows in merge panel indicate co-localization between P-mCherry signal and anti-N protein staining in fixed M compartment axons at 4 h post infection . (C) RABV N protein in SCG cell bodies (CB) at 24 h post axonal infection. Protein lysates were separated using SDS-PAGE, and N protein levels were determined by western blotting. Symbols indicate the presence (+) or absence (-) of RABV infection in N.(TIF)Click here for additional data file.S4 Fig(A) Quantification of % infected cell bodies at 24 h post axonal infection in the absence or presence of 10 μM, 50 μM, or 100 μM emetine in N. (B) Quantification of % infected cell bodies at 24 h post direct S compartment infection in the absence or presence of 100 μM emetine in N. Emetine was added to N, 1 h prior to infection in S. Emetine was washed out at 5 hpi. Black dots represent individual tri-chambers. Horizontal lines and error bars represent mean ± SD with **p = 0.004, ****p < 0.0001 using one-way ANOVA (ns = not significant using unpaired t-test).(TIF)Click here for additional data file.S5 Fig(A) Brightfield images of cell bodies and axons at 24 h after a 6 h treatment with emetine (100 μM) or vehicle in N . Emetine was washed out of the N compartment at 6 h post treatment, and fresh media was added. (B) Experimental setup for live/dead SYTOX cell assay: DiI was added to the N compartment axons to label connected cell bodies in red. Emetine was added to the N compartment for 6 h (or to the S compartment for 24 h as a positive control for cell death). Emetine was washed out of axons after 6 h. At 6 or 24 h, cell bodies were stained with SYTOX green nucleic acid stain (5 nM) for 10 min and imaged. Representative images show cell bodies in the S compartment at 6 h after emetine treatment in N and 24 h after emetine treatment in S . (C) Table indicates percentage of dead cell bodies at 6 h or 24 h after a 6 h emetine treatment in N versus a 6 h or 24 h emetine treatment in S. The percentage of dead cells refers to the percentage of connected cell bodies (DiI-positive) that are stained with SYTOX .(TIF)Click here for additional data file.S6 Fig(A) Experimental setup for entry assay. (B) Quantification of % infected cell bodies at 24 hpi when N compartment axons were incubated with RABV inoculum for 1 to 300 minutes. The virus inoculum was removed following the designated incubation period, and the axons were washed three times with PBS to remove extracellular particles. Black dots represent individual tri-chambers. Horizontal lines and error bars represent mean ± SD with *p = 0.02, ****p < 0.0001 using one-way ANOVA (ns = not significant).(TIF)Click here for additional data file.


The chemical structures of the new and known compounds were unambiguously elucidated based on HRESIMS and exhaustive 1D and 2D NMR spectral analysis as well as a comparison of their NMR data with those of jasplakinolide (1). The isolated jasplakinolides inhibited the growth of mouse lymphoma (L5178Y) cells in vitro with IC50 values in the low micromolar to nanomolar range.A new acylic jasplakinolide congener ( The UV spectrum of 4 revealed an additional absorption maximum (λmax) at 310.7 nm to the usual maximal absorption wavelengths at 220 and 280 nm found for other derivatives, indicating the presence of an acyclic jasplakinolide congener. Based on the determined molecular formula and UV spectrum of 4 and by comparison to the previously reported jasplakinolides, it was suggested to be the reported derivative (+)-jasplakinolide Z5 +  and m/z 763.2311 [M + Na]+ .5(+)-Jasplakinolide Z (4): white amorphous solid; +58.0° ; UV (MeOH) λmax 220, 280, 310.7 nm; 1H-NMR and 13-C NMR see m/z 758.2749 [M + NH4]+  and m/z 763.2306 [M + Na]+ .Cytotoxicity was tested against L5178Y mouse lymphoma cells using the microculture tetrazolium (MTT) assay as described earlier ,19. All