Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA

ABSTRACT The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.

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Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production

Journal of Virology ◽

10.1128/jvi.00627-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 8178-8189 ◽

Cited By ~ 134

Author(s):

Matthew F. McCown ◽

Andrew Pekosz

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Reverse Genetics ◽

Infectious Virus ◽

Virus Assembly ◽

Virus Production ◽

Cytoplasmic Tail ◽

M2 Protein ◽

Virus Particles ◽

M1 Protein

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.

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Influenza A Virus Expresses High Levels of an Unusual Class of Small Viral Leader RNAs in Infected Cells

mBio ◽

10.1128/mbio.00204-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 52

Author(s):

Jennifer L. Umbach ◽

Hui-Ling Yen ◽

Leo L. M. Poon ◽

Bryan R. Cullen

Keyword(s):

Influenza Virus ◽

Life Cycle ◽

Influenza A Virus ◽

Influenza A ◽

Viral Life Cycle ◽

Virus Species ◽

Genomic Rna ◽

Pathogenic Potential ◽

Human Virus ◽

Very High

ABSTRACTEvidence has recently accumulated suggesting that small noncoding RNAs, and particularly microRNAs, have the potential to strongly affect the replication and pathogenic potential of a range of human virus species. Here, we report the use of deep sequencing to comprehensively analyze small viral RNAs (18 to 27 nucleotides [nt]) produced during infection by influenza A virus. Although influenza A virus differs from most other RNA viruses in that it replicates its genome in the nucleus and is therefore exposed to the nuclear microRNA processing factors Drosha and DGCR8, we did not observe any microRNAs encoded by influenza virus genes. However, influenza virus infection did induce the expression of very high levels—over 100,000 copies per cell by 8 h postinfection—of a population of 18- to 27-nt small viral leader RNAs (leRNAs) that originated from the precise 5′ ends of all eight influenza virus genomic RNA (vRNA) segments. Like the vRNAs themselves, our data indicate that the leRNAs also bear a 5′-terminal triphosphate and are therefore not capable of functioning as microRNAs. Instead, the high-level production of leRNAs may imply a role in another aspect of the viral life cycle, such as regulation of the switch from viral mRNA transcription to genomic RNA synthesis.IMPORTANCEInfluenza A virus is an important human pathogen that has the potential to give rise to serious pandemics. Here, we demonstrate that influenza A virus induces the expression of very high levels of small viral leader RNAs (leRNAs) within hours of infection. These RNAs are unusual in that they bear a 5′ triphosphate and originate from the very 5′ ends of the eight viral genomic RNA (vRNA) segments. Their high expression may imply an important role in the viral life cycle that could potentially serve as a novel target for antiviral drugs.

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Mutations in the Influenza A Virus M1 Protein Enhance Virus Budding To Complement Lethal Mutations in the M2 Cytoplasmic Tail

Journal of Virology ◽

10.1128/jvi.00858-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 5

Author(s):

Hsuan Liu ◽

Michael L. Grantham ◽

Andrew Pekosz

Keyword(s):

Virus Particle ◽

Influenza A Virus ◽

Influenza A ◽

Infectious Virus ◽

Virus Assembly ◽

Virus Production ◽

Cytoplasmic Tail ◽

Filament Formation ◽

Virus Budding ◽

Virus Particles

ABSTRACTThe influenza A virus M1 and M2 proteins play important roles in virus assembly and in the morphology of virus particles. Mutations in the distal cytoplasmic tail region of M2, and in particular a tyrosine-to-alanine mutation at residue 76 (Y76A), were essential for infectious virus production and filament formation while having limited effects on total virus particle budding. Using a novel selection method, mutations at seven different M1 amino acids (residue 73, 94, 135, 136, or 138 or a double mutation, 93/244) that are not found in circulating influenza virus strains or have not been previously identified to play a role in influenza A virus assembly were found to complement the lethal M2Y76A mutation. These M1 suppressor mutations restored infectious virus production in the presence of M2Y76A and mediated increased budding and filament formation even in the absence of M2. However, the efficiency of infectious virus replication was still dependent on the presence of the distal region of the M2 cytoplasmic tail. The data suggest that influenza A virus budding and genome incorporation can occur independently and provide further support for complementary roles of the M1 and M2 proteins in virus assembly.IMPORTANCEInfluenza virus particle assembly involves the careful coordination of various viral and host factors to optimally produce infectious virus particles. We have previously identified a mutation at position 76 of the influenza A virus M2 protein that drastically reduces infectious virus production and filament formation with minimal effects on virus budding. In this work, we identified suppressor mutations in the M1 protein which complement this lethal M2 mutation by increasing the efficiency with which virus particles bud from infected cells and restoring filament formation at the infected-cell surface. M2 distal cytoplasmic domain sequences were still required for optimal infectivity. This indicates that M1 and M2 can functionally replace each other in some, but not all, aspects of virus particle assembly.

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cis-Acting Packaging Signals in the Influenza Virus PB1, PB2, and PA Genomic RNA Segments

Journal of Virology ◽

10.1128/jvi.79.16.10348-10355.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10348-10355 ◽

Cited By ~ 114

Author(s):

Yuying Liang ◽

Ying Hong ◽

Tristram G. Parslow

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Virus Assembly ◽

Virus Genome ◽

Coding Region ◽

Coding Sequences ◽

Viral Packaging ◽

Cis Acting ◽

Virus Particles ◽

Coding Regions

ABSTRACT The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5′ and 3′ untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5′ and 66 bases of 3′ coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5′ coding region but is independent of coding sequences at the 3′ end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5′ and 3′ ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.

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Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology

Journal of Virology ◽

10.1128/jvi.00592-15 ◽

2015 ◽

Vol 89 (17) ◽

pp. 8957-8966 ◽

Cited By ~ 53

Author(s):

Petr Chlanda ◽

Oliver Schraidt ◽

Susann Kummer ◽

James Riches ◽

Heike Oberwinkler ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Electron Tomography ◽

Virus Assembly ◽

Viral Proteins ◽

Particle Release ◽

Virus Like Particles ◽

Virus Particles ◽

M Proteins ◽

Associated Proteins

ABSTRACTThe assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.IMPORTANCEInfluenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

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Friend or Foe: The Role of the Cytoskeleton in Influenza A Virus Assembly

Viruses ◽

10.3390/v11010046 ◽

2019 ◽

Vol 11 (1) ◽

pp. 46 ◽

Cited By ~ 8

Author(s):

Sukhmani Bedi ◽

Akira Ono

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Negative Impact ◽

Virus Assembly ◽

Virus Particles ◽

Cellular Processes ◽

Cellular Machinery ◽

Cytoskeletal Elements ◽

Cytoskeletal System

Influenza A Virus (IAV) is a respiratory virus that causes seasonal outbreaks annually and pandemics occasionally. The main targets of the virus are epithelial cells in the respiratory tract. Like many other viruses, IAV employs the host cell’s machinery to enter cells, synthesize new genomes and viral proteins, and assemble new virus particles. The cytoskeletal system is a major cellular machinery, which IAV exploits for its entry to and exit from the cell. However, in some cases, the cytoskeleton has a negative impact on efficient IAV growth. In this review, we highlight the role of cytoskeletal elements in cellular processes that are utilized by IAV in the host cell. We further provide an in-depth summary of the current literature on the roles the cytoskeleton plays in regulating specific steps during the assembly of progeny IAV particles.

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Faculty Opinions recommendation of Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717961036.793464000 ◽

2012 ◽

Author(s):

Richard Webby

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Functional Characterization

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Influenza A Virus Hemagglutinin and Other Pathogen Glycoprotein Interactions with NK Cell Natural Cytotoxicity Receptors NKp46, NKp44, and NKp30

Viruses ◽

10.3390/v13020156 ◽

2021 ◽

Vol 13 (2) ◽

pp. 156

Author(s):

Jasmina M. Luczo ◽

Sydney L. Ronzulli ◽

Stephen M. Tompkins

Keyword(s):

Influenza Virus ◽

Nk Cells ◽

Influenza A Virus ◽

Influenza A ◽

Nk Cell ◽

Influenza Virus Infection ◽

Target Cells ◽

Natural Cytotoxicity ◽

Activating Receptors ◽

Natural Cytotoxicity Receptors

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions.

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Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus

Viruses ◽

10.3390/v13020234 ◽

2021 ◽

Vol 13 (2) ◽

pp. 234

Author(s):

Sarah Al-Beltagi ◽

Cristian Alexandru Preda ◽

Leah V. Goulding ◽

Joe James ◽

Juan Pu ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Influenza A Virus ◽

Broad Spectrum ◽

Influenza A ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Virus Challenge ◽

2009 H1n1 ◽

Syncytial Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

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