A conserved influenza A virus nucleoprotein code controls specific viral genome packaging

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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Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P 2

Virology ◽

10.1016/j.virol.2016.05.018 ◽

2016 ◽

Vol 496 ◽

pp. 116-126 ◽

Cited By ~ 7

Author(s):

Michinori Kakisaka ◽

Kazunori Yamada ◽

Akiko Yamaji-Hasegawa ◽

Toshihide Kobayashi ◽

Yoko Aida

Keyword(s):

Viral Genome ◽

Influenza A ◽

Viral Rna ◽

Genome Packaging ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region

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vRNA-vRNA interactions in influenza A virus HA vRNA packaging

10.1101/2020.01.15.907295 ◽

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.

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Local structural changes of the influenza A virus ribonucleoprotein complex by single mutations in the specific residues involved in efficient genome packaging

Virology ◽

10.1016/j.virol.2019.03.004 ◽

2019 ◽

Vol 531 ◽

pp. 126-140 ◽

Cited By ~ 4

Author(s):

Naoki Takizawa ◽

Yoshitoshi Ogura ◽

Yoko Fujita ◽

Takeshi Noda ◽

Hideki Shigematsu ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Structural Changes ◽

Genome Packaging ◽

Ribonucleoprotein Complex

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The Surface-Exposed PA51-72-Loop of the Influenza A Virus Polymerase Is Required for Viral Genome Replication

Journal of Virology ◽

10.1128/jvi.00687-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 2

Author(s):

Benjamin E. Nilsson-Payant ◽

Jane Sharps ◽

Narin Hengrung ◽

Ervin Fodor

Keyword(s):

Influenza A Virus ◽

Viral Genome ◽

Influenza A ◽

Replication Initiation ◽

Genome Replication ◽

Influenza A Viruses ◽

Viral Polymerase ◽

Virus Rna ◽

Endonuclease Domain

ABSTRACTThe heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2, and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context, the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited inde novoreplication initiationin vitro. Furthermore, viral RNA (vRNA) synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilization of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular.IMPORTANCEInfluenza A viruses are a major global health threat, not only causing significant morbidity and mortality every year but also having the potential to cause severe pandemic outbreaks like the 1918 influenza pandemic. The viral polymerase is a protein complex which is responsible for transcription and replication of the viral genome and therefore is an attractive target for antiviral drug development. For that purpose it is important to understand the mechanisms of how the virus replicates its genome and how the viral polymerase works on a molecular level. In this report, we characterize the role of the flexible surface-exposed PA51-72-loop in polymerase function and offer new insights into the replication mechanism of influenza A viruses.

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Incorporation of influenza A virus genome segments does not absolutely require wild-type sequences

Journal of General Virology ◽

10.1099/vir.0.010355-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1734-1740 ◽

Cited By ~ 26

Author(s):

Ken Fujii ◽

Makoto Ozawa ◽

Kiyoko Iwatsuki-Horimoto ◽

Taisuke Horimoto ◽

Yoshihiro Kawaoka

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Virus Genome ◽

Wild Type ◽

Genome Packaging ◽

Viral Rnas ◽

Virion Incorporation ◽

Specific Sequences ◽

Type Sequence

The efficient incorporation of influenza virus genome segments into virions is mediated by cis-acting regions at both ends of the viral RNAs. It was shown previously that nt 16–26 at the 3′ end of the non-structural (NS) viral RNA of influenza A virus are important for efficient virion incorporation and that nt 27–56 also contribute to this process. To understand further the signalling requirements for genome packaging, this study performed linker-scanning mutagenesis in the latter region and found that nt 27–35 made an appreciable contribution to the efficient incorporation of the NS segment. An NS vRNA library was then generated composed of an RNA population with randomized nucleotides at positions 16–35 such that the virus could select the sequences it required for virion incorporation. The sequences selected differed from the wild-type sequence and no conserved nucleotides were selected. The ability of non-wild-type sequences to function in this manner indicates that the incorporation of influenza A virus genome segments does not absolutely require specific sequences.

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Importance of the 1+7 configuration of ribonucleoprotein complexes for influenza A virus genome packaging

Nature Communications ◽

10.1038/s41467-017-02517-w ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 22

Author(s):

Takeshi Noda ◽

Shin Murakami ◽

Sumiho Nakatsu ◽

Hirotaka Imai ◽

Yukiko Muramoto ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Virus Genome ◽

Genome Packaging ◽

Ribonucleoprotein Complexes

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

Journal of Virology ◽

10.1128/jvi.01634-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11869-11879 ◽

Cited By ~ 109

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.

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The Human Interferon-Induced MxA Protein Inhibits Early Stages of Influenza A Virus Infection by Retaining the Incoming Viral Genome in the Cytoplasm

Journal of Virology ◽

10.1128/jvi.02220-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 13053-13058 ◽

Cited By ~ 64

Author(s):

Han Xiao ◽

Marian J. Killip ◽

Peter Staeheli ◽

Richard E. Randall ◽

David Jackson

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Viral Genome ◽

Influenza A ◽

Cellular Response ◽

A549 Cells ◽

Human Interferon ◽

Antiviral State ◽

Viral Spread ◽

Influenza A Virus Infection

The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.

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