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

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

Journal of Virology ◽

10.1128/jvi.80.1.252-261.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 252-261 ◽

Cited By ~ 50

Author(s):

John F. Regan ◽

Yuying Liang ◽

Tristram G. Parslow

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Virus Assembly ◽

Functional Characterization ◽

Genomic Rna ◽

Viral Mrnas ◽

Virus Particles ◽

Essential Components ◽

Viral Genomic Rna

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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Phylogenetic and variability study on all known hemagglutinin subtypes of influenza A virus

Bio-Algorithms and Med-Systems ◽

10.1515/bams-2017-0009 ◽

2017 ◽

Vol 13 (3) ◽

Author(s):

Rafał Filip ◽

Jacek Leluk

Keyword(s):

Phylogenetic Analysis ◽

Influenza Virus ◽

Life Cycle ◽

Influenza A Virus ◽

Influenza A ◽

Current Knowledge ◽

Influenza Viruses ◽

Surface Glycoprotein ◽

Long Period ◽

Variability Study

AbstractHemagglutinin (HA) is a surface glycoprotein found in influenza viruses. This particle plays two crucial functions in the viral life cycle: it allows for the attachment of the virus into the host cell and participates in the fusion of the virus and host membranes. There are 18 different subtypes of HA. Recently, the H17 and H18 strains have been discovered whose hosts were bats. The evolution of these two strains had most likely occurred in isolation for a long period of time. This work presents the phylogenetic analysis and study on mutational variability based on sequences from all 18 currently known HA strains belonging to influenza virus type A. The results have been presented regarding the current knowledge about influenza. The classical software (Clustal, PHYLIP, and ConSurf) as well as original applications (SSSSg and Consensus Constructor) have been used in this research.

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Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture

Journal of Virology ◽

10.1128/jvi.01411-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10347-10358 ◽

Cited By ~ 19

Author(s):

Xiangwu Ju ◽

Yiwu Yan ◽

Qiang Liu ◽

Ning Li ◽

Miaomiao Sheng ◽

...

Keyword(s):

Cell Death ◽

Influenza Virus ◽

Life Cycle ◽

Membrane Proteins ◽

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Early Stage ◽

H5n1 Influenza Virus ◽

H5n1 Influenza

ABSTRACTAs a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death.IMPORTANCEThe integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian influenza virus infections at early stage.

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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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STUDIES ON INFLUENZA VIRUS

Journal of Experimental Medicine ◽

10.1084/jem.73.5.581 ◽

1941 ◽

Vol 73 (5) ◽

pp. 581-599 ◽

Cited By ~ 18

Author(s):

Edwin H. Lennette ◽

Frank L. Horsfall

Keyword(s):

Influenza Virus ◽

Chick Embryo ◽

Influenza A Virus ◽

Influenza A ◽

Complement Fixation ◽

Soluble Antigen ◽

Swine Virus ◽

Specific Fixation ◽

Mouse Lungs

Influenza complement fixation tests designed for use with ferret serum are described. Complement-fixing antigens derived from influenza ferret lungs were unsatisfactory due to their low content of soluble antigen; those prepared from mouse lungs or developing chick embryo membranes proved to be better antigenically and were reliable when the various reagents in the test were properly adjusted to eliminate non-specific fixation of complement. The results of cross complement fixation tests indicated that the soluble antigens of the PR8 and W.S. strains of influenza A virus were closely similar, if not identical. They indicated also that the soluble antigen of swine virus possessed components present in the antigens of the human strains of virus.

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Development of Neuraminidase Subtype-Specific Reference Antisera by Recombinant Protein Expressed in Baculovirus

Clinical and Vaccine Immunology ◽

10.1128/cvi.00385-12 ◽

2012 ◽

Vol 20 (2) ◽

pp. 140-145 ◽

Cited By ~ 3

Author(s):

Kyu-Jun Lee ◽

Jun-Gu Choi ◽

Hyun-Mi Kang ◽

Kwang-Il Kim ◽

Choi-Kyu Park ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A Virus ◽

Diagnostic Tests ◽

Influenza A ◽

Recombinant Baculovirus ◽

Cross Reactivity ◽

Specific Reference ◽

Simple Method ◽

Avian Influenza A Virus

ABSTRACTOutbreaks of avian influenza A virus infection, particularly the H5N1 strains that have affected birds and some humans for the past 15 years, have highlighted the need for increased surveillance and disease control. Such measures require diagnostic tests to detect and characterize the different subtypes of influenza virus. In the current study, a simple method for producing reference avian influenza virus antisera to be used in diagnostic tests was developed. Antisera of nine avian influenza A virus neuraminidases (NA) used for NA subtyping were produced using a recombinant baculovirus. The recombinant NA (rNA) proteins were expressed in Sf9 insect cells and inoculated intramuscularly into specific-pathogen-free chickens with the ISA70 adjuvant. The NA inhibition antibody titers of the rNA antiserum were in the ranges of 5 to 8 and 6 to 9 log2units after the primary and boost immunizations, respectively. The antisera were subtype specific, showing low cross-reactivity against every other NA subtype using the conventional thiobarbituric acid NA inhibition assay. These results suggest that this simple method for producing reference NA antisera without purification may be useful for the diagnosis and surveillance of influenza virus.

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REACTIONS BETWEEN INFLUENZA VIRUS AND A COMPONENT OF ALLANTOIC FLUID

Journal of Experimental Medicine ◽

10.1084/jem.88.4.463 ◽

1948 ◽

Vol 88 (4) ◽

pp. 463-484 ◽

Cited By ~ 22

Author(s):

Paul H. Hardy ◽

Frank L. Horsfall

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Allantoic Fluid ◽

Partial Dissociation

Evidence is presented which shows that there is a component present in normal allantoic fluid, probably mucoprotein in nature, capable of combining with influenza A virus (PR8), and that following combination between this component and the virus only partial dissociation of the complex occurs. Evidence is also presented which strongly suggests that the component is present in virus-infected allantoic fluid in which it is in part combined with the virus and in part free although altered by viral action. The probability that the component is present as well in highly purified preparations of influenza virus, and its effect upon various reactions obtained with this agent are discussed.

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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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HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit

Journal of Virology ◽

10.1128/jvi.01896-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 12

Author(s):

Huan Chen ◽

Yingjuan Qian ◽

Xin Chen ◽

Zhiyang Ruan ◽

Yuetian Ye ◽

...

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Molecular Mechanisms ◽

Polymerase Activity ◽

Negative Regulator ◽

Host Protein ◽

Spectrometric Analysis ◽

Rna Polymerase Activity

ABSTRACT The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication. IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation of α-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

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