Viral RNA Polymerase Activity Research Articles

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

Development and Validation of a High-Performance Liquid Chromatography with Tandem Mass Spectrometry (HPLC-MS/MS) Method for Quantification of Major Molnupiravir Metabolite (β-D-N4-hydroxycytidine) in Human Plasma.

Molnupiravir is an antiviral drug against viral RNA polymerase activity approved by the FDA for the treatment of COVID-19, which is metabolized to β-D-N4-hydroxycytidine (NHC) in human blood plasma. A novel method was developed and validated for quantifying NHC in human plasma within the analytical range of 10-10,000 ng/mL using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) to support pharmacokinetics studies. For sample preparation, the method of protein precipitation by acetonitrile was used, with promethazine as an internal standard. Chromatographic separation was carried out on a Shim-pack GWS C18 (150 mm × 4.6 mm, 5 μm) column in a gradient elution mode. A 0.1% formic acid solution in water with 0.08% ammonia solution (eluent A, v/v) and 0.1% formic acid solution in methanol with 0.08% ammonia solution mixed with acetonitrile in a 4:1 ratio (eluent B, v/v) were used as a mobile phase. Electrospray ionization (ESI) was used as an ionization source. The developed method was validated in accordance with the Eurasian Economic Union (EAEU) rules, based on the European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines for the following parameters and used within the analytical part of the clinical study of molnupiravir drugs: selectivity, suitability of standard sample, matrix effect, calibration curve, accuracy, precision, recovery, lower limit of quantification (LLOQ), carryover, and stability.

Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection.

Real-time monitoring of enzyme-catalyzed phosphoribosylation of anti-influenza prodrug favipiravir by time-lapse nuclear magnetic resonance spectroscopy.

Favipiravir (brand name Avigan), a widely known anti-influenza prodrug, is metabolized by endogenous enzymes of host cells to generate the active form, which exerts inhibition of viral RNA-dependent RNA polymerase activity; first, favipiravir is converted to its phosphoribosylated form, favipiravir-ribofuranosyl-5'-monophosphate (favipiravir-RMP), by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Because this phosphoribosylation reaction is the rate-determining step in the generation of the active metabolite, quantitative and real-time monitoring of the HGPRT-catalyzed reaction is essential to understanding the pharmacokinetics of favipiravir. However, assay methods enabling such monitoring have not been established. 19 F- or 31 P-based nuclear magnetic resonance (NMR) are powerful techniques for observation of intermolecular interactions, chemical reactions, and metabolism of molecules of interest, given that NMR signals of the heteronuclei sensitively reflect changes in the chemical environment of these moieties. Here, we demonstrated direct, sensitive, target-selective, nondestructive, and real-time observation of HGPRT-catalyzed conversion of favipiravir to favipiravir-RMP by performing time-lapse 19 F-NMR monitoring of the fluorine atom of favipiravir. In addition, we showed that 31 P-NMR can be used for real-time observation of the identical reaction by monitoring phosphorus atoms of the phosphoribosyl group of favipiravir-RMP and of the pyrophosphate product of that reaction. Furthermore, we demonstrated that NMR approaches permit the determination of general parameters of enzymatic activity such as Vmax and Km . This method not only can be widely employed in enzyme assays, but also may be of use in the screening and development of new favipiravir-analog antiviral prodrugs that can be phosphoribosylated more efficiently by HGPRT, which would increase the intracellular concentration of the drug's active form. The techniques demonstrated in this study would allow more detailed investigation of the pharmacokinetics of fluorinated drugs, and might significantly contribute to opening new avenues for widespread pharmaceutical studies.

Upregulation of viral RNA polymerase activity promotes adaptation of SSPE virus to neuronal cells

Subacute sclerosing panencephalitis (SSPE) is a rare progressive neurodegenerative disease caused by measles virus variants (SSPE viruses) that results in eventual death. Amino acid substitution(s) in the viral fusion (F) protein are key for viral propagation in the brain in a cell-to-cell manner, a specific trait of SSPE viruses, leading to neuropathogenicity. In this study, we passaged an SSPE virus in cultured human neuronal cells and isolated an adapted virus that propagated more efficiently in neuronal cells and exhibited increased cell-to-cell fusion. Contrary to our expectation, the virus harbored mutations in the large protein, a viral RNA-dependent RNA polymerase, and in the phosphoprotein, its co-factor, rather than in the F protein. Our results imply that upregulated RNA polymerase activity, which increases F protein expression and cell-to-cell fusion, could be a viral factor that provides a growth advantage and contributes to the adaptation of SSPE viruses to neuronal cells.

G Protein Subunit β1 Facilitates Influenza A Virus Replication by Promoting the Nuclear Import of PB2.

G protein subunit β1 (GNB1), the beta subunit of the G protein family, plays an important role in regulating transmembrane signal transduction. Although a recent study has demonstrated that GNB1 can bind the matrix protein 1 (M1) to facilitate M1 transport to budding sites and promote the release of progeny influenza A virus (IAV), whether the GNB1 protein has other functions in IAV replication requires further study. Here, we found that GNB1 promoted IAV replication, as virus yield decreased in GNB1 knockdown or knockout cells. GNB1 interacted with polymerase subunits PB2, PB1, and PA. Overexpressed GNB1 facilitated PB2 binding to importin α3, α5, and α7 promoting the nuclear import of PB2, enhancing viral RNA synthesis and polymerase activity. Altogether, our results demonstrated that GNB1 positively regulates virus replication by interacting with polymerase subunits and facilitating the nuclear import of PB2, which provide novel insights into the molecular mechanism of IAV. IMPORTANCE Until now, there has been only one article on the role of GNB1 in IAV budding. No study has investigated the role of GNB1 in IAV replication. In this study, our research demonstrated that GNB1 could increase the interaction between PB2 and the importin α isoform and mediate the nuclear import of PB2. Therefore, GNB1 could promote viral replication and transcription. Our results provide a better understanding of the molecular mechanisms of viral replication and provide potential antiviral drug targets.

From the Editor’s Desk...

From the Editor’s Desk...

IL-26 inhibits hepatitis C virus replication in hepatocytes

Interleukin-26 (IL-26) is a proinflammatory cytokine that has properties atypical for a cytokine, such as direct antibacterial activity and DNA-binding capacity. We previously observed an accumulation of IL-26 in fibrotic and inflammatory lesions in the livers of patients with chronic HCV infection and showed that infiltrating CD3+ lymphocytes were the principal source of IL-26. Surprisingly, IL-26 was also detected in the cytoplasm of hepatocytes from HCV-infected patients, even though these cells do not produce IL-26, even when infected with HCV. Based on this observation and possible interactions between IL-26 and nucleic acids, we investigated the possibility that IL-26 controlled HCV infection independently of the immune system. We evaluated the ability of IL-26 to interfere with HCV replication in hepatocytes and investigated the mechanisms by which IL-26 exerts its antiviral activity. We showed that IL-26 penetrated HCV-infected hepatocytes, where it interacted directly with HCV double-stranded RNA replication intermediates, thereby inhibiting viral replication. IL-26 interfered with viral RNA-dependent RNA polymerase activity, preventing the de novo synthesis of viral genomic single-stranded RNA. These findings reveal a new role for IL-26 in direct protection against HCV infection, independently of the immune system, and increase our understanding of the antiviral defense mechanisms controlling HCV infection. Future studies should evaluate the possible use of IL-26 for treating other chronic disorders caused by RNA viruses, for which few treatments are currently available, or emerging RNA viruses. This study sheds new light on the body's arsenal for controlling hepatitis C virus (HCV) infection and identifies interleukin-26 (IL-26) as an antiviral molecule capable of blocking HCV replication. IL-26, which has unique biochemical and structural characteristics, penetrates infected hepatocytes and interacts directly with viral RNA, thereby blocking viral replication. IL-26 is, therefore, a new player in antiviral defenses, operating independently of the immune system. It is of considerable potential interest for treating HCV infection and other chronic disorders caused by RNA viruses for which few treatments are currently available, and for combating emerging RNA viruses.

In Vitro Replication Inhibitory Activity of Xanthorrhizol against Severe Acute Respiratory Syndrome Coronavirus 2

In spite of the large number of repositioned drugs and direct-acting antivirals in clinical trials for the management of the ongoing COVID-19 pandemic, there are few cost-effective therapeutic options for severe acute respiratory syndrome (SARS) coronavirus 2 (SCoV2) infection. In this paper, we show that xanthorrhizol (XNT), a bisabolane-type sesquiterpenoid compound isolated from the Curcuma xanthorrhizza Roxb., a ginger-line plant of the family Zingiberaceae, displays a potent antiviral efficacy in vitro against SCoV2 and other related coronaviruses, including SARS-CoV-1 (SCoV1) and a common cold-causing human coronavirus. XNT reduced infectious SCoV2 titer by ~3-log10 at 20 μM and interfered with the replication of the SCoV1 subgenomic replicon, while it had no significant antiviral effects against hepatitis C virus and noroviruses. Further, XNT exerted similar antiviral functions against SCoV2 variants, such as a GH clade strain and a delta strain currently predominant worldwide. Neither SCoV2 entry into cells nor the enzymatic activity of viral RNA polymerase (Nsp12), RNA helicase (Nsp13), or the 3CL main protease (Nsp5) was inhibited by XNT. While its CoV replication inhibitory mechanism remains elusive, our results demonstrate that the traditional folk medicine XNT could be a promising antiviral candidate that inhibits a broad range of SCoV2 variants of concern and other related CoVs.

GS-441524 inhibits African swine fever virus infection in vitro

African swine fever virus (ASFV) is a highly infectious and lethal swine pathogen that causes serious socio-economic consequences in endemic countries for which no safe and effective vaccine is currently available. GS-441524, a 1-cyano-substituted adenine C-nucleoside ribose analogue, inhibits viral RNA transcription by competing with natural nucleosides (ATP, TTP, CTP, and GTP) and effectively inhibits viral RNA-dependent RNA polymerase activity. However, whether GS-441524 can inhibit the replication of DNA viruses is unknown. In this study, we confirmed that GS-441524 inhibits ASFV infection in porcine alveolar macrophages (PAMs) in a dose-dependent manner; GS-441524 significantly inhibited ASFV replication at different time points after ASFV infection, particularly at the early stages of viral replication. Notably, GS-441524 did not increase the levels of antiviral cytokines or ATP in PAMs. However, an increase in the concentration of natural ATP in PAMs promoted the replication of ASFV and attenuated the inhibitory effect of GS-441524 in a dose-dependent manner. Our results suggest that GS-441524 is an effective antiviral against ASFV.

Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity.

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.).

Cellular CARD11 Inhibits the Fusogenic Activity of Newcastle Disease Virus via CBM Signalosome-Mediated Furin Reduction in Chicken Fibroblasts.

Newcastle disease virus (NDV) causes an infectious disease that poses a major threat to poultry health. Our previous study identified a chicken brain-specific caspase recruitment domain-containing protein 11 (CARD11) that was upregulated in chicken neurons and inhibited NDV replication. This raises the question of whether CARD11 plays a role in inhibiting viruses in non-neural cells. Here, chicken fibroblasts were used as a non-neural cell model to investigate the role. CARD11 expression was not significantly upregulated by either velogenic or lentogenic NDV infection in chicken fibroblasts. Viral replication was decreased in DF-1 cells stably overexpressing CARD11, while viral growth was significantly increased in the CARD11-knockdown DF-1 cell line. Moreover, CARD11 colocalized with the viral P protein and aggregated around the fibroblast nucleus, suggesting that an interaction existed between CARD11 and the viral P protein; this interaction was further examined by suppressing viral RNA polymerase activity by using a minigenome assay. Viral replication was inhibited by CARD11 in fibroblasts, and this result was consistent with our previous report in chicken neurons. Importantly, CARD11 was observed to reduce the syncytia induced by either velogenic virus infection or viral haemagglutinin-neuraminidase (HN) and F cotransfection in fibroblasts. We found that CARD11 inhibited the expression of the host protease furin, which is essential for cleavage of the viral F protein to trigger fusogenic activity. Furthermore, the CARD11-Bcl10-MALT1 (CBM) signalosome was found to suppress furin expression, which resulted in a reduction in the cleavage efficiency of the viral F protein to further inhibit viral syncytia. Taken together, our findings mainly demonstrated a novel CARD11 inhibitory mechanism for viral fusogenic activity in chicken fibroblasts, and this mechanism explains the antiviral roles of this molecule in NDV pathogenesis.

The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses.

Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3Dpol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens.IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens.

The Impacts of Reassortant Avian Influenza H5N2 Virus NS1 Proteins on Viral Compatibility and Regulation of Immune Responses.

Avian influenza virus (AIV) can cause severe diseases in poultry worldwide. H6N1 AIV was the dominant enzootic subtype in 1985 in the chicken farms of Taiwan until the initial outbreak of a low pathogenic avian influenza (LPAI) H5N2 virus in 2003; thereafter, this and other LPAIs have been sporadically detected. In 2015, the outbreak of three novel H5Nx viruses of highly pathogenic avian influenza (HPAI) emerged and devastated Taiwanese chicken and waterfowl industries. The mechanism of variation in pathogenicity among these viruses is unclear; but, in light of the many biological functions of viral non-structural protein 1 (NS1), including interferon (IFN) antagonist and host range determinant, we hypothesized that NS genetic diversity contributes to AIV pathogenesis. To determine the impact of NS1 variants on viral infection dynamics, we established a reverse genetics system with the genetic backbone of the enzootic Taiwanese H6N1 for generation of reassortant AIVs carrying exogenous NS segments of three different Taiwanese H5N2 strains. We observed distinct cellular distributions of NS1 among the reassortant viruses. Moreover, exchange of the NS segment significantly influenced growth kinetics and induction of cytokines [IFN-α, IFN-β, and tumor necrosis factor alpha (TNF-α)] in an NS1- and host-specific manner. The impact of NS1 variants on viral replication appears related to their synergic effects on viral RNA-dependent RNA polymerase activity and IFN response. With these approaches, we revealed that NS1 is a key factor responsible for the diverse characteristics of AIVs in Taiwan.

Anthralin Suppresses the Proliferation of Influenza Virus by Inhibiting the Cap-Binding and Endonuclease Activity of Viral RNA Polymerase.

Influenza virus RNA-dependent RNA polymerase (vRdRp) does not have capping activity and relies on the capped RNAs produced by the host RNA polymerase II (RNAPII). The viral polymerases process the capped RNAs to produce short capped RNA fragments that are used as primers to initiate the transcription of viral mRNAs. This process, known as cap-snatching, can be targeted by antiviral therapeutics. Here, anthralin was identified as an inhibitor against influenza a virus (IAV) infection by targeting the cap-snatching activity of the viral polymerase. Anthralin, an FDA-approved drug used in the treatment of psoriasis, shows antiviral activity against IAV infection in vitro and in vivo. Importantly, anthralin significantly reduces weight loss, lung injury, and mortality caused by IAV infection in mice. The mechanism of action study revealed that anthralin inhibits the cap-binding function of PB2 subunit and endonuclease activity of PA. As a result, viral mRNA transcription is blocked, leading to the decreases in viral RNA replication and viral protein expression. In conclusion, anthralin has been demonstrated to have the potential of an alternative antiviral against influenza virus infection. Also, targeting the captive pocket structure that includes the N-terminus of PA endonuclease domain and the C-terminal of PB2 cap-binding domain of IAV RdRp may be an excellent strategy for developing anti-influenza drugs.

The Caenorhabditis elegans RIG-I Homolog DRH-1 Mediates the Intracellular Pathogen Response upon Viral Infection.

Mammalian retinoic acid-inducible gene I (RIG-I)-like receptors detect viral double-stranded RNA (dsRNA) and 5'-triphosphorylated RNA to activate the transcription of interferon genes and promote antiviral defense. The Caenorhabditis elegans RIG-I-like receptor DRH-1 promotes defense through antiviral RNA interference (RNAi), but less is known about its role in regulating transcription. Here, we describe a role for DRH-1 in directing a transcriptional response in C. elegans called the intracellular pathogen response (IPR), which is associated with increased pathogen resistance. The IPR includes a set of genes induced by diverse stimuli, including intracellular infection and proteotoxic stress. Previous work suggested that the proteotoxic stress caused by intracellular infections might be the common trigger of the IPR, but here, we demonstrate that different stimuli act through distinct pathways. Specifically, we demonstrate that DRH-1/RIG-I is required for inducing the IPR in response to Orsay virus infection but not in response to other triggers like microsporidian infection or proteotoxic stress. Furthermore, DRH-1 appears to be acting independently of its known role in RNAi. Interestingly, expression of the replication-competent Orsay virus RNA1 segment alone is sufficient to induce most of the IPR genes in a manner dependent on RNA-dependent RNA polymerase activity and on DRH-1. Altogether, these results suggest that DRH-1 is a pattern recognition receptor that detects viral replication products to activate the IPR stress/immune program in C. elegansIMPORTANCEC. elegans lacks homologs of most mammalian pattern recognition receptors, and how nematodes detect pathogens is poorly understood. We show that the C. elegans RIG-I homolog DRH-1 mediates the induction of the intracellular pathogen response (IPR), a novel transcriptional defense program, in response to infection by the natural C. elegans viral pathogen Orsay virus. DRH-1 appears to act as a pattern recognition receptor to induce the IPR transcriptional defense program by sensing the products of viral RNA-dependent RNA polymerase activity. Interestingly, this signaling role of DRH-1 is separable from its previously known role in antiviral RNAi. In addition, we show that there are multiple host pathways for inducing the IPR, shedding light on the regulation of this novel transcriptional immune response.

Development and Characterization of a Reverse-Genetics System for Influenza D Virus.

Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse-genetics system (RGS) is urgently needed, but to date, no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven-plasmid-based system and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV minigenome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein (RNP) complex, resulting in either attenuated or replication-incompetent viruses. Finally, by using the minigenome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3' conserved noncoding region in IDV and influenza C virus (ICV) resulted in the inefficient cross-recognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reverse-genetics system that can be used to further study replication, tropism, and pathogenesis of IDV.IMPORTANCE Influenza D virus (IDV) is a new type of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Increased outbreaks in pigs and serological and genetic evidence of human infection have raised concerns about potential IDV adaptation in humans. Here, we have developed a plasmid-based IDV reverse-genetics system that can generate infectious viruses with replication kinetics similar to those of wild-type viruses following transfection of cultured cells. Further characterization demonstrated that viruses rescued from the described RGS resembled the parental viruses in biological and receptor-binding properties. We also developed and validated an IDV minireplicon reporter system that specifically measures viral RNA polymerase activity. In summary, the reverse-genetics system and minireplicon reporter assay described in this study should be of value in identifying viral determinants of cross-species transmission and pathogenicity of novel influenza D viruses.

Tolerability of Erythrocyte Ribavirin Triphosphate Concentrations Depends on the ITPA Genotype.

Ribavirin (RBV) is an antiviral drug that is part of the current standard therapy for chronic hepatitis C (CHC). It is enzymatically converted to ribavirin triphosphate (RTP) that inhibits the activity of viral RNA polymerase, thereby preventing viral replication. However, one of its adverse effects includes hemolytic anemia that limits its application. The variant of ITPA (inosine triphosphatase), which dephosphorylates inosine triphosphate to inosine monophosphate, is a protective factor for RBV-induced anemia. RTP is an important metabolite required for ribavirin action. This study evaluated the time-dependent association of RTP concentrations in erythrocytes, RBV-induced toxicity, and virological response to RBV treatment for hepatitis C. A total of 28 Japanese patients with CHC were treated with RBV/peg-interferon/simeprevir or RBV/sofosbuvir and were genotyped for ITPA variants (rs1127354 and rs7270101). We measured RTP concentrations in erythrocytes in a total of 76 samples collected at 4, 8, and 12 weeks from the initiation of treatment. The ITPA rs1127354 variant was found in 7 patients. This was associated with significantly higher RTP concentrations in erythrocytes than in the wild-type patients (P < 0.001). Moreover, a significant correlation was observed between RTP concentrations and decline in hemoglobin (Hb) levels from baseline values in ITPA wild type and rs1127354 variant 12 weeks after treatment initiation (P < 0.01; r = -0.618 and -0.967, respectively). Multiple regression analysis revealed that ITPA genotype and erythrocyte RTP concentrations were major factors associated with reduced Hb levels in RBV therapy for CHC. However, we did not find any association between erythrocyte concentrations and virological response. The increased tolerability to RTP concentrations in erythrocytes in the ITPA variant rs1127354 plays a role in preventing RBV-induced severe anemia in this ITPA variant.

Avian Influenza A Virus Polymerase Recruits Cellular RNA Helicase eIF4A3 to Promote Viral mRNA Splicing and Spliced mRNA Nuclear Export.

The influenza A virus replicates in a broad range of avian and mammalian species by hijacking cellular factors and processes. Avian influenza A viruses (AIVs) generally propagated poorly in mammalian cells, but some mutants of virus-encoded RNA polymerase components, especially PB2 subunit, can overcome host restriction. Host factors associated with PB2 may be essential for efficient AIV replication in mammalian cells. Here, we infected human cells with the PB2 Flag-tagged replication-competent recombinant AIV and identified cellular proteins that coprecipitate with PB2 protein by mass spectrometry. We confirmed one of the coprecipitating host factors, DEAD-box protein eIF4A3, that interacts with viral PB2, PB1, and NP proteins. Depletion of endogenous eIF4A3 significantly reduced virus replication. Later studies showed that eIF4A3 is essential for viral RNA polymerase activity and viral RNAs synthesis. Upon systematic dissection of the influenza virus progeny mRNA generation, from pre-mRNA processing to nuclear export, we found that the depletion of eIF4A3 resulted in significant defects in the ratio of M2 to M1 and NS2 to NS1, and the proportion of viral spliced mRNA in the nucleus increased, indicating that eIF4A3 plays a significant function in viral nascent intron mRNA splicing and spliced mRNA (M2 and NS2) nuclear export. Additionally, we confirmed that in specific deletion of eIF4A3, the synthesis of reduced NS2 can significantly impair neo-synthetized viral ribonucleoprotein (vRNP) nuclear export. Taken together, our findings revealed that eIF4A3 is a key mediator of AIV polymerase activity, mRNA splicing, and spliced mRNA nuclear export.

HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit.

The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication.IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation of α-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.