scholarly journals Temperature-Sensitive Lesions in Two Influenza A Viruses Defective for Replicative Transcription Disrupt RNA Binding by the Nucleoprotein

1999 ◽  
Vol 73 (9) ◽  
pp. 7349-7356 ◽  
Author(s):  
Liz Medcalf ◽  
Emma Poole ◽  
Debra Elton ◽  
Paul Digard
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Point Mutations ◽  
Temperature Shift ◽  
Binding Activity ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Coding Region ◽  
Influenza A Viruses

ABSTRACT The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30°C. This suggests that the RNA-binding activity of NP is required for replicative transcription.

scholarly journals Characterization of the 1918 “Spanish” Influenza Virus Matrix Gene Segment

2002 ◽  
Vol 76 (21) ◽  
pp. 10717-10723 ◽  
Author(s):  
Ann H. Reid ◽  
Thomas G. Fanning ◽  
Thomas A. Janczewski ◽  
Sherman McCall ◽  
Jeffery K. Taubenberger
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Transmembrane Domain ◽  
Phylogenetic Analyses ◽  
Gene Segment ◽  
High Yield ◽  
Coding Region ◽  
Matrix Gene ◽  
Virus Strains

ABSTRACT The coding region of influenza A virus RNA segment 7 from the 1918 pandemic virus, consisting of the open reading frames of the two matrix genes M1 and M2, has been sequenced. While this segment is highly conserved among influenza virus strains, the 1918 sequence does not match any previously sequenced influenza virus strains. The 1918 sequence matches the consensus over the M1 RNA-binding domains and nuclear localization signal and the highly conserved transmembrane domain of M2. Amino acid changes that correlate with high yield and pathogenicity in animal models were not found in the 1918 strain. Phylogenetic analyses suggest that both genes were mammalian adapted and that the 1918 sequence is very similar to the common ancestor of all subsequent human and classical swine matrix segments. The 1918 sequence matches other mammalian strains at 4 amino acids in the extracellular domain of M2 that differ consistently between avian and mammalian strains, suggesting that the matrix segment may have been circulating in human strains for at least several years before 1918.

scholarly journals Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes

2003 ◽  
Vol 77 (19) ◽  
pp. 10575-10583 ◽  
Author(s):  
Tokiko Watanabe ◽  
Shinji Watanabe ◽  
Takeshi Noda ◽  
Yutaka Fujii ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Influenza Viruses ◽  
Coding Region ◽  
Influenza A Viruses ◽  
Virion Incorporation ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Genomic Regions

ABSTRACT At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3′ end of the coding region and 80 nucleotides at the 5′ end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.

scholarly journals NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Aitor Nogales ◽  
Luis Martinez-Sobrido ◽  
David J. Topham ◽  
Marta L. DeDiego
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Immune Responses ◽  
Influenza A ◽  
Innate Immune ◽  
Temperature Sensitive ◽  
Ns1 Protein ◽  
Innate Immune Responses ◽  
Influenza A Viruses ◽  
Virus Surveillance

ABSTRACT Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo.

scholarly journals Structure of an H3N2 influenza virus nucleoprotein

2021 ◽  
Vol 77 (7) ◽  
Author(s):  
Michael L. Knight ◽  
Haitian Fan ◽  
David L. V. Bauer ◽  
Jonathan M. Grimes ◽  
Ervin Fodor ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Binding ◽  
H3n2 Virus ◽  
Influenza A Viruses ◽  
Seasonal Epidemic ◽  
H3n2 Influenza ◽  
Structural Levels ◽  
Viral Genomic Rna ◽  
H3n2 Influenza Virus

Influenza A viruses of the H1N1 and H3N2 subtypes are responsible for seasonal epidemic events. The influenza nucleoprotein (NP) binds to the viral genomic RNA and is essential for its replication. Efforts are under way to produce therapeutics and vaccines targeting the NP. Despite this, no structure of an NP from an H3N2 virus has previously been determined. Here, the structure of the A/Northern Territory/60/1968 (H3N2) influenza virus NP is presented at 2.2 Å resolution. The structure is highly similar to those of the A/WSN/1933 (H1N1) and A/Hong Kong/483/97 (H5N1) NPs. Nonconserved amino acids are widely dispersed both at the sequence and structural levels. A movement of the 73–90 RNA-binding loop is observed to be the key difference between the structure determined here and previous structures. The data presented here increase the understanding of structural conservation amongst influenza NPs and may aid in the design of universal interventions against influenza.

scholarly journals Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

2018 ◽  
Vol 5 (3) ◽  
pp. 76 ◽  
Author(s):  
Jessica Benkaroun ◽  
Gregory Robertson ◽  
Hugh Whitney ◽  
Andrew Lang
Keyword(s):  
Influenza Virus ◽  
Host Species ◽  
Influenza A ◽  
Wild Bird ◽  
Coding Region ◽  
Influenza A Viruses ◽  
Protein Coding ◽  
Coding Regions ◽  
Negative Sense ◽  
Specific Sequences

The genomes of influenza A viruses (IAVs) comprise eight negative-sense single-stranded RNA segments. In addition to the protein-coding region, each segment possesses 5′ and 3′ non-coding regions (NCR) that are important for transcription, replication and packaging. The NCRs contain both conserved and segment-specific sequences, and the impacts of variability in the NCRs are not completely understood. Full NCRs have been determined from some viruses, but a detailed analysis of potential variability in these regions among viruses from different host groups and locations has not been performed. To evaluate the degree of conservation in NCRs among different viruses, we sequenced the NCRs of IAVs isolated from different wild bird host groups (ducks, gulls and seabirds). We then extended our study to include NCRs available from the National Center for Biotechnology Information (NCBI) Influenza Virus Database, which allowed us to analyze a wider variety of host species and more HA and NA subtypes. We found that the amount of variability within the NCRs varies among segments, with the greatest variation found in the HA and NA and the least in the M and NS segments. Overall, variability in NCR sequences was correlated with the coding region phylogeny, suggesting vertical coevolution of the (coding sequence) CDS and NCR regions.

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 Replication-incompetent influenza A viruses that stably express a foreign gene

2011 ◽  
Vol 92 (12) ◽  
pp. 2879-2888 ◽  
Author(s):  
Makoto Ozawa ◽  
Sylvia T. Victor ◽  
Andrew S. Taft ◽  
Shinya Yamada ◽  
Chengjun Li ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Reporter Gene ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Influenza Viruses ◽  
Foreign Gene ◽  
Coding Region ◽  
Influenza A Viruses ◽  
Gene Encoding ◽  
Virus Research

A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.

Occurrence of temperature-sensitive influenza A viruses in nature.

1982 ◽  
Vol 41 (2) ◽  
pp. 353-359 ◽  
Author(s):  
C M Chu ◽  
S F Tian ◽  
G F Ren ◽  
Y M Zhang ◽  
L X Zhang ◽  
...  
Keyword(s):  
Influenza A ◽  
Temperature Sensitive ◽  
Influenza A Viruses

scholarly journals Unique structural solution from a VH3-30 antibody targeting the hemagglutinin stem of influenza A viruses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wayne D. Harshbarger ◽  
Derrick Deming ◽  
Gordon J. Lockbaum ◽  
Nattapol Attatippaholkun ◽  
Maliwan Kamkaew ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Influenza A ◽  
Critical Role ◽  
Binding Activity ◽  
Human Antibody ◽  
Broadly Neutralizing Antibodies ◽  
Influenza A Viruses ◽  
Universal Vaccine ◽  
Structural Details ◽  
Structural Solution

AbstractBroadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.

scholarly journals Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model

Vaccines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 40
Author(s):  
Wen-Chun Liu ◽  
Raffael Nachbagauer ◽  
Daniel Stadlbauer ◽  
Shirin Strohmeier ◽  
Alicia Solórzano ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Virus Vaccine ◽  
Antibody Responses ◽  
Upper Respiratory Tract ◽  
Influenza A Viruses ◽  
Humoral And Cellular Immunity ◽  
Stalk Domain ◽  
One Year

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.

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