scholarly journals A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF

2008 ◽  
Vol 82 (13) ◽  
pp. 6419-6426 ◽  
Author(s):  
Qinshan Gao ◽  
Edward W. A. Brydon ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Surface Glycoprotein ◽  
Influenza C Virus ◽  
Major Surface

ABSTRACT Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.

scholarly journals Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

2000 ◽  
Vol 74 (10) ◽  
pp. 4634-4644 ◽  
Author(s):  
Jie Zhang ◽  
Andrew Pekosz ◽  
Robert A. Lamb
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Virus Assembly ◽  
Intrinsic Property ◽  
Influenza Viruses ◽  
Apical Surface ◽  
Cytoplasmic Tails ◽  
Fusion Glycoprotein ◽  
Influenza C Virus ◽  
Lipid Raft Microdomains

ABSTRACT Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2 protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.

scholarly journals Specific Residues of the Influenza A Virus Hemagglutinin Viral RNA Are Important for Efficient Packaging into Budding Virions

2007 ◽  
Vol 81 (18) ◽  
pp. 9727-9736 ◽  
Author(s):  
Glenn A. Marsh ◽  
Raheleh Hatami ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Viral Rna ◽  
Wild Type ◽  
Reading Frame ◽  
Synonymous Mutations ◽  
Green Fluorescent ◽  
Ha Protein ◽  
Virus Replication Cycle

ABSTRACT A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3′ and 80 5′ nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.

scholarly journals Structure and Receptor Binding Preferences of Recombinant Hemagglutinins from Avian and Human H6 and H10 Influenza A Virus Subtypes

2015 ◽  
Vol 89 (8) ◽  
pp. 4612-4623 ◽  
Author(s):  
Hua Yang ◽  
Paul J. Carney ◽  
Jessie C. Chang ◽  
Julie M. Villanueva ◽  
James Stevens
Keyword(s):  
Influenza A Virus ◽  
Receptor Binding ◽  
Influenza A ◽  
Influenza Viruses ◽  
Surface Glycoprotein ◽  
Health Concern ◽  
Glycan Microarray ◽  
Detailed Structural Analysis ◽  
Major Surface ◽  
Human Infections

ABSTRACTDuring 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections.IMPORTANCEHuman infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses.

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 Hemagglutinin stalk domain from H5N1 strain as a potentially universal antigen.

2014 ◽  
Vol 61 (3) ◽  
Author(s):  
Karolina Uranowska ◽  
Jolanta Tyborowska ◽  
Anna Jurek ◽  
Bogusław Szewczyk ◽  
Beata Gromadzka
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Virus Antigen ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Health Concern ◽  
Stalk Domain ◽  
Major Surface

Influenza A virus infections are the major public health concern and cause significant morbidity and mortality each year worldwide. Vaccination is the main strategy of influenza epidemic prevention. However, seasonal vaccines induce strain-specific immunity and must be reformulated annually based on prediction of the strains that will circulate in the next season. Thus, it is essential to develop vaccines that would induce broad and persistent immunity to influenza viruses. Hemagglutinin is the major surface antigen of the influenza virus. Recent studies revealed the importance of HA stalk-specific antibodies in neutralization of different influenza virus strains. Therefore, it is important to design an immunogen that would focus the immune response on the HA stalk domain in order to elicit neutralizing antibodies. In the present study, we report characterization of a conserved truncated protein, potentially a universal influenza virus antigen from the H5N1 Highly Pathogenic Avian Influenza A virus strain. Our results indicate that exposure of the HA stalk domain containing conserved epitopes results in cross reactivity with different antibodies (against group 1 and 2 HAs). Additionally, we conclude that HA stalk domain contains not only conformational epitopes recognized by universal FI6 antibody, but also linear epitopes recognized by other antibodies.

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

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.

scholarly journals Plasmid-only rescue of influenza A virus vaccine candidates

2001 ◽  
Vol 356 (1416) ◽  
pp. 1965-1973 ◽  
Author(s):  
J. H. Schickli ◽  
A. Flandorfer ◽  
T. Nakaya ◽  
L. Martinez-Sobrido ◽  
A. Garcia-Sastre ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Genetic Manipulation ◽  
Influenza Viruses ◽  
High Yield ◽  
Ns1 Protein ◽  
Potential Threat ◽  
Rescue System ◽  
Rapid Generation

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative–stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics–based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid–only rescue system that allows the rapid generation of high–yield recombinant vaccine strains. Second, we propose developing second–generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.

scholarly journals mSphere of Influence: Resolution of the Structure of an Influenza Virus Polymerase Is a Game Changer

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Mathilde Richard
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Structural Insight ◽  
Rna Promoter ◽  
Insight Into ◽  
Virus Biology ◽  
Game Changer

ABSTRACT Mathilde Richard works in the field of virology, more specifically on the evolution and pathogenesis of influenza viruses. In this mSphere of Influence article, she reflects on how the two articles “Structure of Influenza A Polymerase Bound to the Viral RNA Promoter” by A. Pflug, D. Guilligay, S. Reich, and S. Cusack (Nature 516:355–360, 2014, https://doi.org/10.1038/nature14008) and “Structural Insight into Cap-Snatching and RNA Synthesis by Influenza Polymerase” by S. Reich, D. Guilligay, A. Pflug, H. Malet, I. Berger, et al. (Nature 516:361–366, 2014, https://doi.org/10.1038/nature14009) made an impact on her by providing new grounds to study the influenza virus polymerase and its role in virus biology and evolution.

scholarly journals Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells

2005 ◽  
Vol 79 (21) ◽  
pp. 13811-13816 ◽  
Author(s):  
Pascale Massin ◽  
Pierre Rodrigues ◽  
Monica Marasescu ◽  
Sylvie van der Werf ◽  
Nadia Naffakh
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Promoter Sequence ◽  
Influenza Viruses ◽  
Rna Polymerase I ◽  
Influenza A Viruses ◽  
Virus Research ◽  
Influenza Vaccine Production ◽  
Polymerase I

ABSTRACT Reverse genetics techniques to rescue influenza viruses have thus far been based on the use of a human polymerase I (PolI) promoter to direct the synthesis of the eight viral RNAs. They can only be used on cells from primate origin due to the species specificity of the PolI promoter. Here we report the cloning of the chicken PolI promoter sequence and the generation of recombinant influenza virus upon transfection of bidirectional PolI/PolII plasmids in avian cells. Potential contributions of this new reverse genetics system in the fields of influenza virus research and influenza vaccine production are discussed.

scholarly journals Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Jeremy R. Keown ◽  
Zihan Zhu ◽  
Loïc Carrique ◽  
Haitian Fan ◽  
Alexander P. Walker ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Structural Data ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Influenza A Viruses ◽  
Seasonal Epidemics ◽  
1918 Influenza

AbstractInfluenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.

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