Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

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Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

Journal of Virology ◽

10.1128/jvi.02246-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1788-1801 ◽

Cited By ~ 26

Author(s):

Shoko Nakamura ◽

Masayuki Horie ◽

Tomo Daidoji ◽

Tomoyuki Honda ◽

Mayo Yasugi ◽

...

Keyword(s):

Epithelial Cells ◽

Viral Replication ◽

Influenza A Virus ◽

Knockout Mice ◽

Influenza A ◽

Clinical Symptoms ◽

Early Stage ◽

Mucin Production ◽

Infected Cells ◽

Basal Epithelial Cells

ABSTRACTInfluenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained fromgalnt3-knockout mice. Interestingly, IAV-infectedgalnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells.IMPORTANCEViral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.

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Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein

PLoS ONE ◽

10.1371/journal.pone.0031063 ◽

2012 ◽

Vol 7 (2) ◽

pp. e31063 ◽

Cited By ~ 43

Author(s):

Xiaoling Liu ◽

Zhendong Zhao ◽

Chongfeng Xu ◽

Lei Sun ◽

Jilong Chen ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Cyclophilin A ◽

M1 Protein

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Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection

Cell Reports ◽

10.1016/j.celrep.2021.109159 ◽

2021 ◽

Vol 35 (7) ◽

pp. 109159

Author(s):

Xiaoyuan Bai ◽

Wenxian Yang ◽

Xiaohan Luan ◽

Huizi Li ◽

Heqiao Li ◽

...

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Group A Streptococcus ◽

Cyclophilin A ◽

Group A ◽

Influenza A Virus Infection

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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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Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle—Structure, Interactions, and Implications for the Emergence of New Strains

Pathogens ◽

10.3390/pathogens9110951 ◽

2020 ◽

Vol 9 (11) ◽

pp. 951

Author(s):

Julita Piasecka ◽

Aleksandra Jarmolowicz ◽

Elzbieta Kierzek

Keyword(s):

Viral Replication ◽

Influenza A Virus ◽

Rna Structure ◽

Influenza A ◽

Respiratory Infections ◽

Specific Interaction ◽

Replication Cycle ◽

Genome Packaging ◽

Functional Roles ◽

Interaction Sites

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

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Mutations in NA That Induced Low pH-Stability and Enhanced the Replication of Pandemic (H1N1) 2009 Influenza A Virus at an Early Stage of the Pandemic

PLoS ONE ◽

10.1371/journal.pone.0064439 ◽

2013 ◽

Vol 8 (5) ◽

pp. e64439 ◽

Cited By ~ 21

Author(s):

Tadanobu Takahashi ◽

Jiasheng Song ◽

Takashi Suzuki ◽

Yoshihiro Kawaoka

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Early Stage ◽

Low Ph ◽

Ph Stability ◽

Pandemic H1n1 ◽

Pandemic H1n1 2009

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Matrix proteins of enveloped viruses: a case study of Influenza A virus M1 protein

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2018.1436089 ◽

2018 ◽

Vol 37 (3) ◽

pp. 671-690 ◽

Cited By ~ 10

Author(s):

Larisa V. Kordyukova ◽

Eleonora V. Shtykova ◽

Lyudmila A. Baratova ◽

Dmitri I. Svergun ◽

Oleg V. Batishchev

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Matrix Proteins ◽

Enveloped Viruses ◽

M1 Protein

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Chios mastic gum inhibits influenza A virus replication and viral pathogenicity

Journal of General Virology ◽

10.1099/jgv.0.001550 ◽

2021 ◽

Author(s):

Dong-In Kim ◽

Yong-Bin Cho ◽

Younghyun Lim ◽

So-Hee Hong ◽

Bumsuk Hahm ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Bacterial Infections ◽

Early Stage ◽

Antiviral Drug ◽

Virus Infectivity ◽

Viral Attachment ◽

Cellular Processes ◽

Chios Mastic Gum

Chios mastic gum (CMG), a resin of the mastic tree (Pistacia lentiscus var. chia), has been used to treat multiple disorders caused by gastrointestinal malfunctions and bacterial infections for more than 2500 years. However, little is known about CMG’s antiviral activity. CMG is known to influence multiple cellular processes such as cell proliferation, differentiation and apoptosis. As virus replication is largely dependent on the host cellular metabolism, it is conceivable that CMG regulates virus infectivity. Therefore, in this study, we evaluated CMG’s potential as an antiviral drug to treat influenza A virus (IAV) infection. CMG treatment dramatically reduced the cytopathogenic effect and production of RNAs, proteins and infectious particles of IAV. Interestingly, CMG interfered with the early stage of the virus life cycle after viral attachment. Importantly, the administration of CMG greatly ameliorated morbidity and mortality in IAV-infected mice. The results suggest that CMG displays a potent anti-IAV activity by blocking the early stage of viral replication. Thus, mastic gum could be exploited as a novel therapeutic agent against IAV infection.

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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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