The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route

ABSTRACTInfluenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine.IMPORTANCENeuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to viral replication in ducks via the natural route of infection and demonstrate that maintenance of NA activity under low-pH conditions is associated with the biological properties of the virus. These findings provide insights into the mechanisms of replication of influenza A virus in ducks.

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Differential Recognition of Influenza A Viruses by M158–66Epitope-Specific CD8+T Cells Is Determined by Extraepitopic Amino Acid Residues

Journal of Virology ◽

10.1128/jvi.02439-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 1009-1022 ◽

Cited By ~ 15

Author(s):

Carolien E. van de Sandt ◽

Joost H. C. M. Kreijtz ◽

Martina M. Geelhoed-Mieras ◽

Nella J. Nieuwkoop ◽

Monique I. Spronken ◽

...

Keyword(s):

Amino Acid ◽

Influenza A Virus ◽

Influenza A ◽

Rational Design ◽

Amino Acid Residues ◽

Virus Infections ◽

Human Influenza ◽

Influenza A Viruses ◽

Specific Ctls ◽

Naturally Occurring

ABSTRACTNatural influenza A virus infections elicit both virus-specific antibody and CD4+and CD8+T cell responses. Influenza A virus-specific CD8+cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158–66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158–66epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8+T cell recognition of the M158–66epitope. These data indicate that human influenza A viruses can impair recognition by M158–66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses.IMPORTANCEInfluenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8+cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M158–66epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158–66epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

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Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

Journal of Virology ◽

10.1128/jvi.75.17.8127-8136.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8127-8136 ◽

Cited By ~ 78

Author(s):

Daniel R. Perez ◽

Ruben O. Donis

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Amino Acid ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Sequences ◽

Influenza Viruses ◽

Virus Growth ◽

Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

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Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

Journal of General Virology ◽

10.1099/vir.0.010660-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1730-1733 ◽

Cited By ~ 8

Author(s):

Natalia L. Varich ◽

Konstantin S. Kochergin-Nikitsky ◽

Evgeny V. Usachev ◽

Olga V. Usacheva ◽

Alexei G. Prilipov ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Influenza A Virus ◽

Influenza A ◽

Antigenic Variation ◽

Strain Differences ◽

Antigenic Structure ◽

Amino Acid Residues ◽

Mutant Proteins ◽

Antigenic Sites

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

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Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance

Journal of Virology ◽

10.1128/jvi.00161-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 6

Author(s):

Nancy Hom ◽

Lauren Gentles ◽

Jesse D. Bloom ◽

Kelly K. Lee

Keyword(s):

Cell Culture ◽

Influenza Virus ◽

Amino Acid ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Matrix Protein ◽

Influenza Virus Infection ◽

Sequence Diversity ◽

Virus Protein

ABSTRACTInfluenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCEThe M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved protein’s function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1’s tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

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Amino Acid Residues 68–71 Contribute to Influenza A Virus PB1-F2 Protein Stability and Functions

Frontiers in Microbiology ◽

10.3389/fmicb.2017.00692 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Yi-Ying Cheng ◽

Shih-Rang Yang ◽

Ying-Ting Wang ◽

Yu-Hsin Lin ◽

Chi-Ju Chen

Keyword(s):

Amino Acid ◽

Protein Stability ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Residues

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The host factor ANP32A is required for influenza A virus vRNA and cRNA synthesis

10.1101/2021.04.30.442228 ◽

2021 ◽

Author(s):

Benjamin E Nilsson-Payant ◽

Benjamin R. tenOever ◽

Aartjan J.W. te Velthuis

Keyword(s):

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Host Factor ◽

Viral Rna ◽

Molecular Evidence ◽

Influenza A Viruses ◽

Ribonucleoprotein Complexes ◽

Rna Genome

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is bound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the complementary RNA (cRNA), and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases and differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from a cRNA, but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32 is not only needed for the actively replicating polymerase, but also for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation.

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Key amino acid residues of neuraminidase involved in influenza A virus entry

Pathogens and Disease ◽

10.1093/femspd/ftz063 ◽

2019 ◽

Vol 77 (6) ◽

Author(s):

Fangzhao Chen ◽

Teng Liu ◽

Jiagui Xu ◽

Yingna Huang ◽

Shuwen Liu ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Influenza A Virus ◽

Viral Entry ◽

Influenza A ◽

Mutational Analysis ◽

Critical Role ◽

Target Cells ◽

Amino Acid Residues ◽

Influenza Virus Neuraminidase

ABSTRACT Generally, influenza virus neuraminidase (NA) plays a critical role in the release stage of influenza virus. Recently, it has been found that NA may promote influenza virus to access the target cells. However, the mechanism remain unclear. Here, we reported that peramivir indeed possessed anti-influenza A virus (IAV) activity in the stage of viral entry. Importantly, we verified the critical residues of influenza NA involved in the viral entry. As a result, peramivir as an efficient NA inhibitor could suppress the initiation of IAV infection. Furthermore, mutational analysis showed NA might be associated with viral entry via amino acids residues R118, E119, D151, R152, W178, I222, E227, E276, R292 and R371. Our results demonstrated NA must contain the key amino acid residues can involve in IAV entry.

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Identification of Amino Acid Residues in Influenza A Virus PA-X That Contribute to Enhanced Shutoff Activity

Frontiers in Microbiology ◽

10.3389/fmicb.2019.00432 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 5

Author(s):

Kohei Oishi ◽

Seiya Yamayoshi ◽

Yoshihiro Kawaoka

Keyword(s):

Amino Acid ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Residues

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Amino Acid Residues in the Fusion Peptide Pocket Regulate the pH of Activation of the H5N1 Influenza Virus Hemagglutinin Protein

Journal of Virology ◽

10.1128/jvi.02238-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3568-3580 ◽

Cited By ~ 74

Author(s):

Mark L. Reed ◽

Hui-Ling Yen ◽

Rebecca M. DuBois ◽

Olga A. Bridges ◽

Rachelle Salomon ◽

...

Keyword(s):

Amino Acid ◽

Influenza A Virus ◽

Influenza A ◽

Coiled Coil ◽

Fusion Peptide ◽

Influenza Viruses ◽

Amino Acid Residues ◽

H5n1 Influenza ◽

Coiled Coil Domain ◽

Ha Protein

ABSTRACT The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.

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