scholarly journals Influenza A Viruses with Mutations in the M1 Helix Six Domain Display a Wide Variety of Morphological Phenotypes

2005 ◽  
Vol 79 (2) ◽  
pp. 1262-1270 ◽  
Author(s):  
Laura M. Burleigh ◽  
Lesley J. Calder ◽  
John J. Skehel ◽  
David A. Steinhauer
Keyword(s):  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Reverse Genetics ◽  
Virus Assembly ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Influenza A Viruses ◽  
Binding Function ◽  
Wide Range

ABSTRACT Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated “helix six.” This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.

scholarly journals Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine

2012 ◽  
Vol 93 (10) ◽  
pp. 2204-2214 ◽  
Author(s):  
Lindomar Pena ◽  
Amy L. Vincent ◽  
Crystal L. Loving ◽  
Jamie N. Henningson ◽  
Kelly M. Lager ◽  
...  
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Viruses ◽  
Dependent Manner ◽  
Lung Pathology ◽  
Influenza A Viruses ◽  
Virus Shedding ◽  
The Impact ◽  
Strain Dependent

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

scholarly journals Antigenic drift in the evolution of H1N1 influenza A viruses resulting from deletion of a single amino acid in the haemagglutinin gene

2007 ◽  
Vol 88 (12) ◽  
pp. 3209-3213 ◽  
Author(s):  
Natalie J. McDonald ◽  
Catherine B. Smith ◽  
Nancy J. Cox
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Reverse Genetics ◽  
Selective Advantage ◽  
H1n1 Influenza ◽  
Antigenic Drift ◽  
Surface Glycoprotein ◽  
Single Amino Acid ◽  
Influenza A Viruses ◽  
Sequence Comparisons

Two genetically distinct lineages of H1N1 influenza A viruses, circulated worldwide before 1994, were antigenically indistinguishable. In 1994, viruses emerged in China, including A/Beijing/262/95, with profound antigenic differences from the contemporary circulating H1N1 strains. Haemagglutinin sequence comparisons of either a predecessor virus, A/Hebei/52/94, or one representative of the cocirculating A/Bayern/7/95-like clade, A/Shenzhen/227/95, revealed a deletion of K at position 134 (H3 numbering) in the antigenic variants. The K134 deletion conferred a selective advantage to the Chinese deletion lineage, such that it eventually gave rise to currently circulating H1 viruses. Using reverse genetics to generate viruses with either an insertion or deletion of aa 134, we have confirmed that the K134 deletion, rather than a constellation of sublineage specific amino acid changes, was sufficient for the antigenic difference observed in the Chinese deletion lineage, and reinsertion of K134 revealed the requirement of a compatible neuraminidase surface glycoprotein for viral growth.

scholarly journals Asymmetric structure of the influenza A virus and novel function of the matrix protein M1

2016 ◽  
Vol 61 (4) ◽  
pp. 149-154
Author(s):  
O. P. Zhirnov
Keyword(s):  
Influenza Virus ◽  
Virus Particle ◽  
Target Cell ◽  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Structural Function ◽  
Asymmetric Structure ◽  
Bipolar Structure

Influenza virus is an enveloped virus. It comprises two major modules: external lipoprotein envelope and internal ribonucleoprotein (RNP) containing the genomic negative-strand RNA. Lipoprotein envelope contains four vital proteins: hemagglutinin (HA), neuraminidase (NA), transmembrane ionic channel M2, and minor amounts of nuclear export protein NEP. RNP contains RNA and four polypeptides: major nucleocapsid protein NP and three polymerase subunits PB1, PB2, PA. Both modules are linked with each other by matrix M1 maintaining the virus integrity. According to the structural function, NP and M1 are predominant in virus particle in the amounts of 1000 and 3000 molecules, respectively. In addition to the structural function, M1 plays a role in regulation of intracellular and nuclear migration of viral RNP and virus assembly, referred as budding process, at the plasma membrane in infected cells. The bipolar structure of the influenza virus characterized by asymmetric location of RNP and nonregular distribution of M1 and M2 inside the virion is reviewed. The role of M1 in maintaining the asymmetric structure of the virus particle and regulation of RNP transport inside virus particle is considered. First experimental data confirming (i) intravirion RNP transport and its outside exit directed by the M1 and (ii) the importance of this process in virus uncoating and initiation of infection in target cell are discussed. A novel class of antiviral agents activating ATP-ase of the early endosome compartment in the target cell is discussed.

scholarly journals Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells

2014 ◽  
Vol 89 (1) ◽  
pp. 300-311 ◽  
Author(s):  
Shijuan Gao ◽  
Jiaoxiang Wu ◽  
Ran-Yi Liu ◽  
Jiandong Li ◽  
Liping Song ◽  
...  
Keyword(s):  
Viral Replication ◽  
Influenza A Virus ◽  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Molecular Mechanisms ◽  
Attachment Site ◽  
Influenza A Viruses ◽  
Screening Assay ◽  
Range Restriction

ABSTRACTInfluenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathioneS-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication.IMPORTANCEAlthough the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication.

scholarly journals Features of Nuclear Export Signals of NS2 Protein of Influenza D Virus

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1100
Author(s):  
Lingcai Zhao ◽  
Huizhi Xia ◽  
Jingjin Huang ◽  
Yiqing Zheng ◽  
Chang Liu ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Influenza A ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Mammalian Species ◽  
Nucleocytoplasmic Transport ◽  
Influenza A Viruses ◽  
Alanine Scanning Mutagenesis ◽  
The Third ◽  
Ribonucleoprotein Complexes

Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

scholarly journals A Mutant Influenza Virus That Uses an N1 Neuraminidase as the Receptor-Binding Protein

2013 ◽  
Vol 87 (23) ◽  
pp. 12531-12540 ◽  
Author(s):  
Kathryn A. Hooper ◽  
Jesse D. Bloom
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Influenza A ◽  
Binding Pocket ◽  
Binding Activity ◽  
Dependent Manner ◽  
Influenza A Viruses ◽  
Cell Agglutination ◽  
Binding Function ◽  
Receptor Binding Activity

In the vast majority of influenza A viruses characterized to date, hemagglutinin (HA) is the receptor-binding and fusion protein, whereas neuraminidase (NA) is a receptor-cleaving protein that facilitates viral release but is expendable for entry. However, the NAs of some recent human H3N2 isolates have acquired receptor-binding activity via the mutation D151G, although these isolates also appear to retain the ability to bind receptors via HA. We report here the laboratory generation of a mutation (G147R) that enables an N1 NA to completely co-opt the receptor-binding function normally performed by HA. Viruses with this mutant NA grow to high titers even in the presence of extensive mutations to conserved residues in HA's receptor-binding pocket. When the receptor-binding NA is paired with this binding-deficient HA, viral infectivity and red blood cell agglutination are blocked by NA inhibitors. Furthermore, virus-like particles expressing only the receptor-binding NA agglutinate red blood cells in an NA-dependent manner. Although the G147R NA receptor-binding mutant virus that we characterize is a laboratory creation, this same mutation is found in several natural clusters of H1N1 and H5N1 viruses. Our results demonstrate that, at least in tissue culture, influenza virus receptor-binding activity can be entirely shifted from HA to NA.

scholarly journals Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions

2006 ◽  
Vol 80 (5) ◽  
pp. 2318-2325 ◽  
Author(s):  
Yukiko Muramoto ◽  
Ayato Takada ◽  
Ken Fujii ◽  
Takeshi Noda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
...  
Keyword(s):  
Influenza A ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Viral Rna ◽  
Green Fluorescent Protein Gene ◽  
Influenza A Viruses ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Green Fluorescent ◽  
Efficient Viral Replication

ABSTRACT The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.

ViDiT–CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications

2018 ◽  
Vol 64 (10) ◽  
pp. 761-773 ◽  
Author(s):  
Joost T.P. Verhoeven ◽  
Marta Canuti ◽  
Hannah J. Munro ◽  
Suzanne C. Dufour ◽  
Andrew S. Lang
Keyword(s):  
Influenza A ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Quality Data ◽  
Library Preparation ◽  
Virus Discovery ◽  
Influenza A Viruses ◽  
Wide Range ◽  
Functional Profiling ◽  
Biology Laboratory

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (less than US$5 per sample), and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing, and microbiome profiling. ViDiT–CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal–cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT–CACTUS demonstrated its validity in a wide range of microbiology applications, and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

scholarly journals Membrane-tethered mucin-like polypeptides sterically inhibit binding and slow fusion kinetics of influenza A virus

2020 ◽  
Vol 117 (23) ◽  
pp. 12643-12650 ◽  
Author(s):  
Corleone S. Delaveris ◽  
Elizabeth R. Webster ◽  
Steven M. Banik ◽  
Steven G. Boxer ◽  
Carolyn R. Bertozzi
Keyword(s):  
Lipid Bilayers ◽  
Influenza A ◽  
Conformational Transition ◽  
Molecular Model ◽  
High Surface ◽  
Protective Mechanism ◽  
Dependent Manner ◽  
Influenza A Viruses ◽  
Virus Binding ◽  
Kinetics Of

The mechanism(s) by which cell-tethered mucins modulate infection by influenza A viruses (IAVs) remain an open question. Mucins form both a protective barrier that can block virus binding and recruit IAVs to bind cells via the sialic acids of cell-tethered mucins. To elucidate the molecular role of mucins in flu pathogenesis, we constructed a synthetic glycocalyx to investigate membrane-tethered mucins in the context of IAV binding and fusion. We designed and synthesized lipid-tethered glycopolypeptide mimics of mucins and added them to lipid bilayers, allowing chemical control of length, glycosylation, and surface density of a model glycocalyx. We observed that the mucin mimics undergo a conformational change at high surface densities from a compact to an extended architecture. At high surface densities, asialo mucin mimics inhibited IAV binding to underlying glycolipid receptors, and this density correlated to the mucin mimic’s conformational transition. Using a single virus fusion assay, we observed that while fusion of virions bound to vesicles coated with sialylated mucin mimics was possible, the kinetics of fusion was slowed in a mucin density-dependent manner. These data provide a molecular model for a protective mechanism by mucins in IAV infection, and therefore this synthetic glycocalyx provides a useful reductionist model for studying the complex interface of host–pathogen interactions.

scholarly journals Eco-Epidemiological Evidence of the Transmission of Avian and Human Influenza A Viruses in Wild Pigs in Campeche, Mexico

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 528
Author(s):  
Brenda Aline Maya-Badillo ◽  
Rafael Ojeda-Flores ◽  
Andrea Chaves ◽  
Saul Reveles-Félix ◽  
Guillermo Orta-Pineda ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Serum Samples ◽  
Fragmented Habitats ◽  
Wild Pigs ◽  
Wide Range ◽  
Influenza Transmission ◽  
Human Viruses ◽  
Positive Results

Influenza, a zoonosis caused by various influenza A virus subtypes, affects a wide range of species, including humans. Pig cells express both sialyl-α-2,3-Gal and sialyl-α-2,6-Gal receptors, which make them susceptible to infection by avian and human viruses, respectively. To date, it is not known whether wild pigs in Mexico are affected by influenza virus subtypes, nor whether this would make them a potential risk of influenza transmission to humans. In this work, 61 hogs from two municipalities in Campeche, Mexico, were sampled. Hemagglutination inhibition assays were performed in 61 serum samples, and positive results were found for human H1N1 (11.47%), swine H1N1 (8.19%), and avian H5N2 (1.63%) virus variants. qRT-PCR assays were performed on the nasal swab, tracheal, and lung samples, and 19.67% of all hogs were positive to these assays. An avian H5N2 virus, first reported in 1994, was identified by sequencing. Our results demonstrate that wild pigs are participating in the exposure, transmission, maintenance, and possible diversification of influenza viruses in fragmented habitats, highlighting the synanthropic behavior of this species, which has been poorly studied in Mexico.

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