scholarly journals Fundamental contribution and host range determination of ANP32 protein family in influenza A virus polymerase activity

2019 ◽  
Author(s):  
Haili Zhang ◽  
Zhenyu Zhang ◽  
Yujie Wang ◽  
Meiyue Wang ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Viral Polymerase ◽  
Human Influenza Virus ◽  
Virus Rna

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase, and to both influence polymerase activity and interspecies restriction. Here we report that either ANP32A or ANP32B is indispensable for influenza A virus RNA replication. The contribution of ANP32B is equal to that of ANP32A, and together they play a fundamental role in the activity of mammalian influenza A virus polymerase, while neither human ANP32A nor ANP32B support the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving ANP32A alone to support viral replication. Two amino acid mutations at sites 129-130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A&B as key factors for both virus replication and adaption.IMPORTANCEThe key host factors involved in the influenza A viral the polymerase activity and RNA replication remain largely unknown. Here we provide evidence that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza virus RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily non-functional in supporting viral replication because of a 129-130 site mutation. The 129-130 site plays an important role in ANP32A/B and viral polymerase interaction, therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

scholarly journals Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Haili Zhang ◽  
Zhenyu Zhang ◽  
Yujie Wang ◽  
Meiyue Wang ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Cellular Protein ◽  
Human Influenza ◽  
Viral Polymerase

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation.IMPORTANCEThe key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

scholarly journals Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

2010 ◽  
Vol 84 (17) ◽  
pp. 8691-8699 ◽  
Author(s):  
Tatiana Fislová ◽  
Benjamin Thomas ◽  
Katy M. Graef ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Mammalian Cells ◽  
Influenza A ◽  
Mitochondrial Protein ◽  
Host Factors ◽  
Fk506 Binding Protein ◽  
Virus Rna ◽  
Rna Accumulation

ABSTRACT The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), α- and β-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

scholarly journals Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Benjamin E. Nilsson ◽  
Aartjan J. W. te Velthuis ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza A Viruses ◽  
Viral Polymerase ◽  
Virus Rna ◽  
Rna Genome ◽  
Transcription And Replication

ABSTRACT The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

scholarly journals Influenza A Virus RNA Polymerase Has the Ability To Stutter at the Polyadenylation Site of a Viral RNA Template during RNA Replication

1999 ◽  
Vol 73 (6) ◽  
pp. 5240-5243 ◽  
Author(s):  
Hongyong Zheng ◽  
Hye Annie Lee ◽  
Peter Palese ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Viral Polymerase ◽  
Virus Rna ◽  
Independent Synthesis ◽  
New Evidence ◽  
Virus Mrnas

ABSTRACT The viral polymerase of influenza virus, a negative-strand RNA virus, is believed to polyadenylate the mRNAs by stuttering at a stretch of five to seven uridine residues which are located close to the 5′ ends of the viral RNA templates. However, a mechanism of polyadenylation based on a template-independent synthesis of the poly(A) tail has not been excluded. In this report, we present new evidence showing the inherent ability of the viral polymerase to stutter at the poly(U) stretch of a viral RNA template during RNA replication. Variants which possess 1- to 13-nucleotide-long insertions at the poly(U) stretch have been identified. These results support a stuttering mechanism for the polyadenylation of influenza virus mRNAs.

scholarly journals Phylogenetic Evidence against Evolutionary Stasis and Natural Abiotic Reservoirs of Influenza A Virus

2008 ◽  
Vol 82 (7) ◽  
pp. 3769-3774 ◽  
Author(s):  
Michael Worobey
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Human Influenza ◽  
Human Influenza Virus ◽  
Virus Rna ◽  
Evolutionary Stasis

ABSTRACT Zhang et al. (G. Zhang, D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. Rogers, J. Virol. 80:12229-12235, 2006) have claimed to have recovered influenza A virus RNA from Siberian lake ice, postulating that ice might represent an important abiotic reservoir for the persistence and reemergence of this medically important pathogen. A rigorous phylogenetic analysis of these influenza A virus hemagglutinin gene sequences, however, indicates that they originated from a laboratory reference strain derived from the earliest human influenza A virus isolate, WS/33. Contrary to Zhang et al.'s assertions that the Siberian “ice viruses” are most closely related either to avian influenza virus or to human influenza virus strains from Asia from the 1960s (Zhang et al., J. Virol. 81:2538 [erratum], 2007), they are clearly contaminants from the WS/33 positive control used in their laboratory. There is thus no credible evidence that environmental ice acts as a biologically relevant reservoir for influenza viruses. Several additional cases with findings that seem at odds with the biology of influenza virus, including modern-looking avian influenza virus RNA sequences from an archival goose specimen collected in 1917 (T. G. Fanning, R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger, J. Virol. 76:7860-7862, 2002), can also be explained by laboratory contamination or other experimental errors. Many putative examples of evolutionary stasis in influenza A virus appear to be due to laboratory artifacts.

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

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
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.

scholarly journals Rewiring the RNAs of influenza virus to prevent reassortment

2009 ◽  
Vol 106 (37) ◽  
pp. 15891-15896 ◽  
Author(s):  
Qinshan Gao ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Influenza Viruses ◽  
Ha Gene ◽  
Ns Gene ◽  
Virus Rna ◽  
Reassortant Viruses ◽  
Packaging Signals

Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses.

scholarly journals Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation

2015 ◽  
Vol 90 (1) ◽  
pp. 586-590 ◽  
Author(s):  
Stéphanie Anchisi ◽  
Jessica Guerra ◽  
Geneviève Mottet-Osman ◽  
Dominique Garcin
Keyword(s):  
Influenza Virus ◽  
Retinoic Acid ◽  
Complex Formation ◽  
Influenza A Virus ◽  
Rna Structure ◽  
Influenza A ◽  
Double Stranded Rna ◽  
Virus Rna ◽  
Inducible Gene

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-Iin vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.

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

2019 ◽  
Vol 93 (13) ◽  
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.

scholarly journals The mechanism of resistance to favipiravir in influenza

2018 ◽  
Vol 115 (45) ◽  
pp. 11613-11618 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Aartjan J. W. te Velthuis ◽  
Robert A. Fletcher ◽  
Pinky Langat ◽  
Maria Zambon ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
H1n1 Influenza ◽  
Viral Fitness ◽  
Virus Infections ◽  
Virus Rna ◽  
Evolution Of Resistance ◽  
Emergence Of Resistance

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.

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