scholarly journals A synergistic effect between 3′ terminal noncoding and adjacent coding regions of influenza A virus HA segment on template preference

2021 ◽  
Author(s):  
Yue Xiao ◽  
Wenyu Zhang ◽  
Minglei Pan ◽  
David L. V. Bauer ◽  
Yuhai Bi ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Viruses ◽  
Nucleotide Position ◽  
Virus Growth ◽  
Noncoding Regions ◽  
Negative Strand ◽  
Coding Regions ◽  
Growth Phenotype

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding haemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype-specific, varying significantly in sequence and length at both the 3´ and 5´ termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3´-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multi-segment environment. These results, provide novel insights into the role of the 3´-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3´-end H1-ssNCR and observed a synergistic effect between the 3´-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of HA segment on template preference in a multi-segment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

scholarly journals Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus

2008 ◽  
Vol 82 (23) ◽  
pp. 11869-11879 ◽  
Author(s):  
Edward C. Hutchinson ◽  
Martin D. Curran ◽  
Eliot K. Read ◽  
Julia R. Gog ◽  
Paul Digard
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genome Packaging ◽  
Virus Growth ◽  
Virus Particles ◽  
Synonymous Mutations ◽  
Coding Regions

ABSTRACT The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.

scholarly journals Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

2005 ◽  
Vol 79 (6) ◽  
pp. 3766-3774 ◽  
Author(s):  
Ken Fujii ◽  
Yutaka Fujii ◽  
Takeshi Noda ◽  
Yukiko Muramoto ◽  
Tokiko Watanabe ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Noncoding Region ◽  
Coding Region ◽  
Genome Packaging ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Negative Sense ◽  
Incorporation Efficiency

ABSTRACT The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3′ coding region are critical for efficient NS vRNA incorporation and that deletion of the 3′ segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3′ NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3′ end) form a structure that is required for efficient influenza A virus vRNA packaging.

scholarly journals Nucleotide Sequence Requirements at the 5′ End of the Influenza A Virus M RNA Segment for Efficient Virus Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3384-3388 ◽  
Author(s):  
Makoto Ozawa ◽  
Junko Maeda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
Shinji Watanabe ◽  
Hideo Goto ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Viral Rna ◽  
Coding Region ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Sequence Requirements

ABSTRACT The mechanism by which the influenza A virus genome is packaged into virions is not fully understood. The coding and noncoding regions necessary for packaging of the viral RNA segments, except for the M segment, have been identified. Here, we delineate the M segment regions by incorporating a reporter viral RNA into virions and by generating viruses possessing mutations in the regions. We found that, like the other segments, the M segment coding regions are essential for virion incorporation and that the nucleotide length rather than the nucleotide sequence of the 5′ end of the coding region is important.

scholarly journals Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
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.

scholarly journals Influenza A Virus Negative Strand RNA Is Translated for CD8+ T Cell Immunosurveillance

2018 ◽  
Vol 201 (4) ◽  
pp. 1222-1228 ◽  
Author(s):  
Heather D. Hickman ◽  
Jacqueline W. Mays ◽  
James Gibbs ◽  
Ivan Kosik ◽  
Javier G. Magadán ◽  
...  
Keyword(s):  
T Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Cd8 T Cell ◽  
Negative Strand

scholarly journals Gene Constellation of Influenza A Virus Reassortants with High Growth Phenotype Prepared as Seed Candidates for Vaccine Production

PLoS ONE ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. e20823 ◽  
Author(s):  
Andrew A. Fulvini ◽  
Manojkumar Ramanunninair ◽  
Jianhua Le ◽  
Barbara A. Pokorny ◽  
Jennifer Minieri Arroyo ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
High Growth ◽  
Vaccine Production ◽  
Growth Phenotype

scholarly journals Synergistic Effect of the PDZ and p85 -Binding Domains of the NS1 Protein on Virulence of an Avian H5N1 Influenza A Virus

2013 ◽  
Vol 87 (9) ◽  
pp. 4861-4871 ◽  
Author(s):  
S. Fan ◽  
C. A. Macken ◽  
C. Li ◽  
M. Ozawa ◽  
H. Goto ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Influenza A Virus ◽  
Influenza A ◽  
Ns1 Protein ◽  
Binding Domains ◽  
H5n1 Influenza

scholarly journals vRNA-vRNA interactions in influenza A virus HA vRNA packaging

2020 ◽  
Author(s):  
Sho Miyamoto ◽  
Yukiko Muramoto ◽  
Keiko Shindo ◽  
Yoko Fujita ◽  
Takeshi Morikawa ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Packaging Signal ◽  
Genome Packaging ◽  
Coding Regions ◽  
Signal Region ◽  
Rna Interaction ◽  
Negative Sense

AbstractThe genome of the influenza A virus is composed of eight single-stranded negative-sense RNA segments (vRNAs). The eight different vRNAs are selectively packaged into progeny virions. This process likely involves specific interactions among vRNAs via segment-specific packaging signals located in the 3’ and 5’ terminal coding regions of vRNAs. To identify vRNA(s) that interact with hemagglutinin (HA) vRNA during genome packaging, we generated a mutant virus, HA 5m2, which possessed five silent mutations in the 5’ packaging signal region of HA vRNA. The HA 5m2 virus had a specific defect in HA vRNA incorporation, which reduced the viral replication efficiency. After serial passaging in cells, the virus acquired additional mutations in the 5’ terminal packaging signal regions of both HA and PB2 vRNAs. These mutations contributed to recovery of viral growth and packaging efficiency of HA vRNA. A direct RNA-RNA interaction between the 5’ ends of HA and PB2 vRNAs was confirmed in vitro. Our results indicate that direct interactions of HA vRNA with PB2 vRNA via their packaging signal regions are important for selective genome packaging and enhance our knowledge on the emergence of pandemic influenza viruses through genetic reassortment.

scholarly journals Beneficial Effect of Indigo Naturalis on Acute Lung Injury Induced by Influenza a Virus

2020 ◽  
Author(s):  
Peng Tu ◽  
Rong Tian ◽  
Yan Lu ◽  
Yunyi Zhang ◽  
Haiyan Zhu ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Influenza A Virus ◽  
Lung Tissue ◽  
Influenza A ◽  
Influenza Viruses ◽  
Virus Growth ◽  
Indigo Naturalis ◽  
Anti Inflammatory

Abstract Background: Infections induced by influenza viruses, as well as COVID-19 pandemic induced by SARS-CoV-2 led to Acute lung injury (ALI) and multiorgan failure, during which traditional Chinese medicine played an important role in treatment of the pandemic. The study aimed to investigate the effect of indigo naturalis on ALI induced by influenza A virus (IAV) in mice.Method: The anti-influenza and anti-inflammatory properties of aqueous extracts of indigo naturalis (INAE) were evaluated in vitro. BALB/c mice inoculated intranasally with IAV (H1N1) were treated intragastrically with INAE (40, 80 and 160 mg kg-1/d) 2 h later for 4 or 7 days. Animal mortality and lifespan were recorded. Expression of high mobility group box-1 protein (HMGB-1) and toll-like receptor-4 (TLR4) were evaluated through immunohistological staining. Inflammatory cytokines were also monitored by ELISA.Result: INAE inhibited virus growth on Madin-Darby canine kidney (MDCK) cells and decreased nitric oxide (NO) production from lipopolysaccharide (LPS)-stimulated peritoneal macrophage in vitro. The results showed that oral administration of 160 mg/kg of INAE significantly improved the lifespan (P < 0.01) and survival rate of IAV infected mice, improved lung injury and lowered viral replication in lung tissue (P < 0.01). Treatment with INAE (40, 80 and 160 mg/kg) also significantly increased liver weight and liver index (P < 0.05), as well as spleen and thymus weight and organ index at 160 mg/kg (P < 0.05). The expression of HMGB-1 and TLR4 in lung tissue were also suppressed. Treatment with INAE reduced the high levels of interferon α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted factor (RANTES), interferon induced protein-10 (IP-10), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) (P < 0.05), with increased production of interleukin-10 (IL-10) (P < 0.05). The increased myeloperoxidase (MPO) activity and methylene dioxyamphetamine (MDA) level in lung tissues were inhibited by INAE treatment (P < 0.05).Conclusion: The results showed that INae alleviated IAV induced ALI in mice. The effect of INAE might be related with its anti-virus, anti-inflammatory and anti-oxidation properties, which give a hint that indigo naturalis might be effective on respiratory viruses infected acute lung injury or SAR-CoV-2 caused COVID-19.

Sign in / Sign up

Export Citation Format

Share Document