scholarly journals Neddylation of M1 negatively regulates the replication of influenza A virus

2020 ◽  
Vol 101 (12) ◽  
pp. 1242-1250
Author(s):  
Yucen Li ◽  
Wenjia Chai ◽  
Jie Min ◽  
Zhen Ye ◽  
Xiaomei Tong ◽  
...  
Keyword(s):  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Critical Role ◽  
Protein S ◽  
Wild Type ◽  
Post Translational Modification ◽  
The Stability

Post-translational modification plays a critical role in viral replication. Previously we reported that neddylation of PB2 of influenza A virus (IAV) can inhibit viral replication. However, we found that NEDD8 overexpression can still inhibit the replication of PB2 K699R mutant viruses, implying that other viral protein(s) can be neddylated. In this study, we revealed that M1 of IAV can also be modified by NEDD8. We found that the E3 ligase HDM2 significantly promotes M1 neddylation. Furthermore, we identified M1 K187 as the major neddylation site. We generated an IAV M1 K187R mutant (WSN-M1 K187R) and compared the growth of wild-type and mutant viruses in Madin–Darby canine kidney (MDCK) cells. The data showed that the replication of WSN-M1 K187R was more efficient than that of wild-type WSN. More importantly, we observed that overexpression of NEDD8 inhibited the replication of the wild-type WSN more effectively than that of WSN-M1 K187R. In addition, we found that the neddylation-deficient M1 mutant (M1 K187R) had a longer half-life than that of wild-type M1, indicating that the neddylation of M1 reduces stability. Then we performed a viral infection assay and found that WSN-M1 K187R exhibited greater virulence in mice than wild-type WSN, suggesting that the neddylation of M1 reduced IAV replication in vivo. In conclusion, we uncovered that neddylation of M1 by HDM2 negatively regulates the stability of M1, which in turn inhibits viral replication.

scholarly journals Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

2016 ◽  
Vol 113 (42) ◽  
pp. 11931-11936 ◽  
Author(s):  
Wenqian He ◽  
Gene S. Tan ◽  
Caitlin E. Mullarkey ◽  
Amanda J. Lee ◽  
Mannie Man Wai Lam ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Critical Role ◽  
Host Cells ◽  
Viral Glycoprotein ◽  
Effector Functions ◽  
Dependent Cell

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals Favipiravir-resistant influenza A virus shows potential for transmission

2020 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Ada Yan ◽  
Rebecca Frise ◽  
Jie Zhou ◽  
Jennifer Shelley ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Genetic Background ◽  
Influenza A ◽  
Resistance Mutation ◽  
Polymerase Activity ◽  
Resistant Virus ◽  
Wild Type ◽  
Fitness Advantage

AbstractFavipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

scholarly journals Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

2010 ◽  
Vol 84 (16) ◽  
pp. 8072-8084 ◽  
Author(s):  
Sarah E. Hobdey ◽  
Brian J. Kempf ◽  
Benjamin P. Steil ◽  
David J. Barton ◽  
Olve B. Peersen
Keyword(s):  
Amino Acid ◽  
Rna Binding ◽  
Critical Role ◽  
Polymerase Activity ◽  
Structural Basis ◽  
Wild Type ◽  
Elongation Complex ◽  
Virus Growth ◽  
The Stability

ABSTRACT The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.

scholarly journals Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

2015 ◽  
Vol 89 (11) ◽  
pp. 6067-6079 ◽  
Author(s):  
GuanQun Liu ◽  
Hong-Su Park ◽  
Hyun-Mi Pyo ◽  
Qiang Liu ◽  
Yan Zhou
Keyword(s):  
Viral Replication ◽  
Influenza A Virus ◽  
Promoter Region ◽  
Influenza A ◽  
Rna Synthesis ◽  
Nucleotide Composition ◽  
Viral Rna ◽  
Viral Transcription ◽  
Wild Type ◽  
Viral Promoter

ABSTRACTRetinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, usingin vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation.IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.

scholarly journals Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus

2008 ◽  
Vol 82 (23) ◽  
pp. 11869-11879 ◽  
Author(s):  
Edward C. Hutchinson ◽  
Martin D. Curran ◽  
Eliot K. Read ◽  
Julia R. Gog ◽  
Paul Digard
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genome Packaging ◽  
Virus Growth ◽  
Virus Particles ◽  
Synonymous Mutations ◽  
Coding Regions

ABSTRACT The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.

Temperature-Sensitive Mutants of Influenza A Virus: Transfer of the Two Temperature-Sensitive Lesions in the Udorn/72- ts -1A2 Virus to the A/Hong Kong/123/77 (H1N1) Wild-Type Virus

1980 ◽  
Vol 28 (3) ◽  
pp. 792-798
Author(s):  
Brian R. Murphy ◽  
Nanette T. Hosier ◽  
Robert M. Chanock
Keyword(s):  
Hong Kong ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Genetic Interaction ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Virus Transfer

The influenza A/Udorn/72- ts -1A2 virus possesses temperature-sensitive mutations in the genes coding for the P1 and P3 polymerase proteins. It is being evaluated as a donor of its attenuating temperature-sensitive genes to produce recombinant live vaccine strains of epidemic variants of influenza A virus. Transfer of the P1 and P3 genes to two viruses within the H3N2 subtype of influenza A virus (i.e., the A/Victoria/3/75 and A/Alaska/6/77 viruses) conferred on each variant the following properties: (i) 37°C shutoff temperature for plaque formation, (ii) almost complete restriction of viral replication in the lungs, (iii) a 100-fold restriction of viral replication in the nasal turbinates, and (iv) genetic stability after replication in hamsters. This study was undertaken to determine whether the transfer of the two ts -1A2 temperature-sensitive genes into a virus belonging to the H1N1 subtype (i.e., the A/Hong Kong/123/77 virus) would result in a restriction of replication in vitro and in vivo comparable to that observed with the previously studied H3N2 recombinant viruses in hamsters. This was found to be the case. In addition, infection of hamsters with the A/Hong Kong/77- ts -1A2 virus induced significant resistance to infection with wild-type A/Hong Kong/77 virus. Thus, the two ts -1A2 temperature-sensitive genes attenuated influenza A viruses belonging to two distinct subtypes to a specific and predictable level. An unexpected genetic interaction was observed between several A/Hong Kong/77- ts -1A2 segregants bearing the group 5 (P1) temperature-sensitive lesion. One interpretation of these results is that intracistronic complementation occurred between these segregants.

scholarly journals Tyr82 Amino Acid Mutation in PB1 Polymerase Induces an Influenza Virus Mutator Phenotype

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Tadasuke Naito ◽  
Kazumasa Shirai ◽  
Kotaro Mori ◽  
Hidetaka Muratsu ◽  
Hiroshi Ushirogawa ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Viruses ◽  
Viral Rna ◽  
Wild Type ◽  
Mutator Phenotype ◽  
Polymerase Fidelity

ABSTRACT In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single-amino-acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivo. IMPORTANCE Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live attenuated vaccine candidates.

scholarly journals Chimeric Influenza A Viruses with a Functional Influenza B Virus Neuraminidase or Hemagglutinin

2003 ◽  
Vol 77 (17) ◽  
pp. 9116-9123 ◽  
Author(s):  
Astrid Flandorfer ◽  
Adolfo García-Sastre ◽  
Christopher F. Basler ◽  
Peter Palese
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
Type A ◽  
Influenza B Virus ◽  
Influenza B ◽  
Type B ◽  
Wild Type ◽  
B Virus

ABSTRACT Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain.

scholarly journals Alanine substitutions within a linker region of the influenza A virus non-structural protein 1 alter its subcellular localization and attenuate virus replication

2011 ◽  
Vol 92 (8) ◽  
pp. 1832-1842 ◽  
Author(s):  
Wei Li ◽  
James W. Noah ◽  
Diana L. Noah
Keyword(s):  
Virus Infection ◽  
Subcellular Localization ◽  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Apoptotic Pathway ◽  
Linker Region

The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein and an important virulence factor. It is composed of two well-characterized domains linked by a short, but not well crystallographically defined, region of unknown function. To study the possible function of this region, we introduced alanine substitutions to replace the two highly conserved leucine residues at amino acid positions 69 and 77. The mutant L69,77A NS1 protein retained wild-type (WT)-comparable binding capabilities to dsRNA, cleavage and polyadenylation specificity factor 30 and the p85β subunit of PI3K. A mutant influenza A virus expressing the L69,77A NS1 protein was generated using reverse genetics. L69,77A NS1 virus infection induced significantly higher levels of beta interferon (IFN-β) expression in Madin–Darby canine kidney (MDCK) cells compared with WT NS1 virus. In addition, the replication rate of the L69,77A NS1 virus was substantially lower in MDCK cells but not in Vero cells compared with the WT virus, suggesting that the L69,77A NS1 protein does not fully antagonize IFN during viral replication. L69,77A NS1 virus infection was not able to activate the PI3K/Akt anti-apoptotic pathway, suggesting that the mutant NS1 protein may not be localized such that it has access to p85β in vivo during infection, which was supported by the altered subcellular localization pattern of the mutant NS1 compared with WT NS1 after transfection or virus infection. Our data demonstrate that this linker region between the two domains is critical for the functions of the NS1 protein during influenza A virus infection, possibly by determining the protein’s correct subcellular localization.

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