Amino acid sites related to the PB2 subunits of IDV affect polymerase activity

Abstract Background In 2011, a new influenza virus, named Influenza D Virus (IDV), was isolated from pigs, and then cattle, presenting influenza-like symptoms. IDV is one of the causative agents of Bovine Respiratory Disease (BRD), which causes high morbidity and mortality in feedlot cattle worldwide. To date, the molecular mechanisms of IDV pathogenicity are unknown. Recent IDV outbreaks in cattle, along with serological and genetic evidence of IDV infection in humans, have raised concerns regarding the zoonotic potential of this virus. Influenza virus polymerase is a determining factor of viral pathogenicity to mammals. Methods Here we take a prospective approach to this question by creating a random mutation library about PB2 subunit of the IDV viral polymerase to test which amino acid point mutations will increase viral polymerase activity, leading to increased pathogenicity of the virus. Results Our work shows some exact sites that could affect polymerase activities in influenza D viruses. For example, two single-site mutations, PB2-D533S and PB2-G603Y, can independently increase polymerase activity. The PB2-D533S mutation alone can increase the polymerase activity by 9.92 times, while the PB2-G603Y mutation increments the activity by 8.22 times. Conclusion Taken together, our findings provide important insight into IDV replication fitness mediated by the PB2 protein, increasing our understanding of IDV replication and pathogenicity and facilitating future studies.

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Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Journal of General Virology ◽

10.1099/vir.0.061598-0 ◽

2014 ◽

Vol 95 (5) ◽

pp. 1033-1042 ◽

Cited By ~ 5

Author(s):

Blanca García-Barreno ◽

Teresa Delgado ◽

Sonia Benito ◽

Inmaculada Casas ◽

Francisco Pozo ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Influenza A ◽

Point Mutations ◽

Antigenic Site ◽

Single Amino Acid ◽

Antigenic Structure ◽

Human Antibody ◽

2009 H1n1

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283–6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

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Mutations in the PA Protein of Avian H5N1 Influenza Viruses Affect Polymerase Activity and Mouse Virulence

Journal of Virology ◽

10.1128/jvi.01557-17 ◽

2017 ◽

pp. JVI.01557-17 ◽

Cited By ~ 5

Author(s):

Gongxun Zhong ◽

Mai Quynh Le ◽

Tiago J.S. Lopes ◽

Peter Halfmann ◽

Masato Hatta ◽

...

Keyword(s):

Amino Acid ◽

Case Fatality ◽

Polymerase Activity ◽

Influenza Viruses ◽

Viral Polymerase ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Increased Risk ◽

Severe Infections ◽

Viral Determinants

To study the influenza viral determinants of pathogenicity, we characterized two highly pathogenic avian H5N1 influenza viruses isolated in Vietnam in 2012 (A/duck/Vietnam/QT1480/2012; QT1480) and 2013 (A/duck/Vietnam/QT1728/2013; QT1728) and found that the activity of their polymerase complexes differed significantly, even though both viruses were highly pathogenic in mice. Further studies revealed that the PA-S343A/E347D mutations reduced viral polymerase activity and mouse virulence when tested in the genetic background of QT1728 virus. In contrast, the PA-343S/347E mutations increased the polymerase activity of QT1480 and the virulence of a low pathogenic H5N1 influenza virus. The PA-343S residue (which alone increased viral polymerase activity and mouse virulence significantly relative to viral replication complexes encoding PA-343A) is frequently found in H5N1 influenza viruses of several subclades; infection with a virus possessing this amino acid may pose an increased risk to humans.IMPORTANCEH5N1 influenza viruses cause severe infections in humans with a case fatality rate that exceeds 50%. The factors that determine the high virulence of these viruses in humans are not fully understood. Here, we identified two amino acid changes in the viral polymerase PA protein that affect the activity of the viral polymerase complex and virulence in mice. Infection with viruses possessing these amino acid changes may pose an increased risk to humans.

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Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle

Journal of Virology ◽

10.1128/jvi.00053-16 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3694-3707 ◽

Cited By ~ 17

Author(s):

Laura Marcos-Villar ◽

Alejandra Pazo ◽

Amelia Nieto

Keyword(s):

Influenza Virus ◽

Rna Polymerase Ii ◽

Polymerase Activity ◽

Virus Multiplication ◽

Viral Transcription ◽

Chromatin Remodeler ◽

Viral Polymerase ◽

Chromatin Dynamics ◽

Rnap Ii ◽

Cellular Transcription

ABSTRACTInfluenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents the recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that another CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 (histone 3 with a trimethyl group in lysine 4) histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and the production of infectious particles. Despite the dependence of influenza virus on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1–RNAP II association, as well as the parallel degradation of both proteins during infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle.IMPORTANCEAlthough influenza virus is not integrated into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors, such as RNA polymerase II, and with chromatin remodelers, such as CHD6. We identified the association of viral polymerase with another chromatin remodeler, the CHD1 protein, which positively modulated viral polymerase activity, viral RNA transcription, and virus multiplication. Once viral transcription is complete, RNAP II is degraded in infected cells, probably as a virus-induced mechanism to reduce the antiviral response. CHD1 associated with RNAP II and paralleled its degradation during infection with viruses that induce full or reduced degradation. These findings suggest that RNAP II degradation and CHD1 degradation cooperate to reduce the antiviral response.

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Ubiquitination Upregulates Influenza Virus Polymerase Function

Journal of Virology ◽

10.1128/jvi.01829-16 ◽

2016 ◽

Vol 90 (23) ◽

pp. 10906-10914 ◽

Cited By ~ 27

Author(s):

James Kirui ◽

Arindam Mondal ◽

Andrew Mehle

Keyword(s):

Influenza Virus ◽

Life Cycle ◽

Viral Proteins ◽

Polymerase Activity ◽

Viral Gene ◽

Viral Polymerase ◽

Ubiquitin Proteasome Pathway ◽

Virus Life Cycle ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

ABSTRACTThe influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation.IMPORTANCEThe cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

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

Journal of Virology ◽

10.1128/jvi.00174-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 16

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.

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Comprehensive characterization of amino acid positions in protein structures reveals molecular effect of missense variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002660117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28201-28211

Author(s):

Sumaiya Iqbal ◽

Eduardo Pérez-Palma ◽

Jakob B. Jespersen ◽

Patrick May ◽

David Hoksza ◽

...

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Molecular Mechanisms ◽

Amino Acid Level ◽

Protein Structures ◽

Point Mutations ◽

Independent Set ◽

Clinical Genetics ◽

Missense Variants ◽

The Impact

Interpretation of the colossal number of genetic variants identified from sequencing applications is one of the major bottlenecks in clinical genetics, with the inference of the effect of amino acid-substituting missense variations on protein structure and function being especially challenging. Here we characterize the three-dimensional (3D) amino acid positions affected in pathogenic and population variants from 1,330 disease-associated genes using over 14,000 experimentally solved human protein structures. By measuring the statistical burden of variations (i.e., point mutations) from all genes on 40 3D protein features, accounting for the structural, chemical, and functional context of the variations’ positions, we identify features that are generally associated with pathogenic and population missense variants. We then perform the same amino acid-level analysis individually for 24 protein functional classes, which reveals unique characteristics of the positions of the altered amino acids: We observe up to 46% divergence of the class-specific features from the general characteristics obtained by the analysis on all genes, which is consistent with the structural diversity of essential regions across different protein classes. We demonstrate that the function-specific 3D features of the variants match the readouts of mutagenesis experiments for BRCA1 and PTEN, and positively correlate with an independent set of clinically interpreted pathogenic and benign missense variants. Finally, we make our results available through a web server to foster accessibility and downstream research. Our findings represent a crucial step toward translational genetics, from highlighting the impact of mutations on protein structure to rationalizing the variants’ pathogenicity in terms of the perturbed molecular mechanisms.

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Characteristic amino acid changes of influenza A(H1N1)pdm09 virus PA protein enhance A(H7N9) viral polymerase activity

Virus Genes ◽

10.1007/s11262-016-1311-4 ◽

2016 ◽

Vol 52 (3) ◽

pp. 346-353 ◽

Cited By ~ 2

Author(s):

Jun Liu ◽

Feng Huang ◽

Junsong Zhang ◽

Likai Tan ◽

Gen Lu ◽

...

Keyword(s):

Amino Acid ◽

Influenza A ◽

Polymerase Activity ◽

Viral Polymerase ◽

Pdm09 Virus ◽

Influenza A H1n1

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Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells

Journal of Virology ◽

10.1128/jvi.00130-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 5928-5938 ◽

Cited By ~ 24

Author(s):

Benjamin Mänz ◽

Miranda de Graaf ◽

Ramona Mögling ◽

Mathilde Richard ◽

Theo M. Bestebroer ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Avian Influenza ◽

Mammalian Cells ◽

Influenza A ◽

Polymerase Activity ◽

Host Adaptation ◽

Influenza Viruses ◽

Amino Acid Substitutions ◽

Avian Influenza A Virus

ABSTRACTA strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified usingin vitroassays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts.IMPORTANCEInfluenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions—most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2—have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans.

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Adaptation of Borna Disease Virus to New Host Species Attributed to Altered Regulation of Viral Polymerase Activity

Journal of Virology ◽

10.1128/jvi.00334-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7933-7940 ◽

Cited By ~ 16

Author(s):

Andreas Ackermann ◽

Peter Staeheli ◽

Urs Schneider

Keyword(s):

Amino Acid ◽

Disease Virus ◽

Host Species ◽

Polymerase Activity ◽

Viral Polymerase ◽

New Host ◽

Borna Disease Virus ◽

Altered Regulation ◽

Borna Disease ◽

New Host Species

ABSTRACT Borna disease virus (BDV) can persistently infect the central nervous system of a broad range of mammalian species. Mice are resistant to infections with primary BDV isolates, but certain laboratory strains can be adapted to replicate in mice. We determined the molecular basis of adaptation by studying mutations acquired by a cDNA-derived BDV strain during one brain passage in rats and three passages in mice. The adapted virus propagated efficiently in mouse brains and induced neurological disease. Its genome contained seven point mutations, three of which caused amino acid changes in the L polymerase (L1116R and N1398D) and in the polymerase cofactor P (R66K). Recombinant BDV carrying these mutations either alone or in combination all showed enhanced multiplication speed in Vero cells, indicating improved intrinsic viral polymerase activity rather than adaptation to a mouse-specific factor. Mutations R66K and L1116R, but not N1398D, conferred replication competence of recombinant BDV in mice if introduced individually. Virus propagation in mouse brains was substantially enhanced if both L mutations were present simultaneously, but infection remained mostly nonsymptomatic. Only if all three amino acid substitutions were combined did BDV replicate vigorously and induce early disease in mice. Interestingly, the virulence-enhancing effect of the R66K mutation in P could be attributed to reduced negative regulation of polymerase activity by the viral X protein. Our data demonstrate that BDV replication competence in mice is mediated by the polymerase complex rather than the viral envelope and suggest that altered regulation of viral gene expression can favor adaptation to new host species.

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