Structural Basis for RNA Binding and Homo-Oligomer Formation by Influenza B Virus Nucleoprotein

Ng, Adolf K.Y.; Lam, Mandy Ka-Han; Zhang, Hongmin; Wang, Jinhuan; Au, Shannon Wing-Ngor; Chan, Paul K.S.; Wang, Jia-Huai; Shaw, Pang‐Chui

doi:10.1128/jvi.00073-12

Cited by 58 publications

(64 citation statements)

References 32 publications

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“…It is possible that the mutations investigated in this study affected the structure of the protein such that multiple functions of this region of BNP were affected. Unfortunately it is not possible to relate these functions to the structure of the protein, as this region of BNP is highly flexible and does not allow for X-ray crystallographic studies (23). The fact that the N-terminal extension of BNP is completely absent from the NP of influenza A viruses suggests that influenza A and B viruses have evolved different strategies for regulating transcription and replication of their genome.…”

Section: Discussionmentioning

confidence: 99%

“…One of the striking differences between the NP of these viruses is that the first 69 residues of BNP show no homology to those of ANP, and there is an evolutionarily conserved 50-amino-acid extension on the N terminus of BNP that is absent from ANP (14). The molecular structure of BNP recently has been solved, and interestingly, the entire N-terminal extension is missing from this structure due to the lack of electron density, suggesting that this region of BNP is highly flexible (23). Previous work reported that this N-terminal extension is not essential for nuclear accumulation of BNP or for the protein to function in an in vitro transcription/replication assay (14).…”

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confidence: 99%

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The N Terminus of the Influenza B Virus Nucleoprotein Is Essential for Virus Viability, Nuclear Localization, and Optimal Transcription and Replication of the Viral Genome

Sherry

Smith

Davidson

et al. 2014

J Virol

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The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However, NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50-amino-acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension; however, this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. The truncation of more than three amino acids prevented virus recovery, suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in the nuclear localization of BNP, with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first 10 residues of BNP demonstrated few differences in nuclear localization; however, the viruses exhibited significant reductions in viral mRNA transcription and genome replication, resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall, our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP but also for regulating viral mRNA transcription and genome replication. IMPORTANCEThe multifunctional NP of influenza viruses has roles throughout the viral replication cycle; therefore, it is essential for virus viability. Despite high levels of homology between the NP of influenza A and B viruses, the NP of influenza B virus contains an evolutionarily conserved 50-amino-acid N-terminal extension that is absent from the NP of influenza A viruses. In this study, we show that this N-terminal extension is essential for virus viability, and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore, we demonstrate for the first time that the N terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension, these viruses may have evolved separate mechanisms to regulate these processes.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

The N Terminus of the Influenza B Virus Nucleoprotein Is Essential for Virus Viability, Nuclear Localization, and Optimal Transcription and Replication of the Viral Genome

Sherry

Smith

Davidson

et al. 2014

J Virol

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“…For several negative-strand RNA viruses, assembly of the RNP has been shown to involve nucleocapsid protein polymerization to enwrap the entire viral RNA genome (43)(44)(45)(46)(47). Arenavirus nucleoprotein homo-oligomerization, which has been documented for members of both the Old World (LASV and LCMV) and New World (TCRV) groups, has been proposed to be an essential element for arenavirus RNP assembly (11,(23)(24)(25).…”

Section: Discussionmentioning

confidence: 99%

Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

D'antuono

Loureiro

Foscaldi

et al. 2014

J Virol

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The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCEThe mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding. V iruses belonging to the Arenaviridae family are classified into two main groups, Old World (OW) and New World (NW), according to phylogeny, serological properties, and geographical distribution (1). Several members of this family, such as the NW Junin virus (JUNV) and the OW Lassa virus (LASV), produce serious hemorrhagic fever in humans. Tacaribe virus (TCRV), the prototype of NW arenaviruses, is nonpathogenic, closely related to JUNV, and has been used as a model in numerous studies (1-4). Arenaviruses ar...

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“…LASV NP is associated with multiple activities, including RNA binding, cap snatching, and an exonuclease activity related to immunosuppression (17)(18)(19). Lastly, the structure of the influenza virus NP is different from any of these NPs described above but is conserved within the Orthomyxoviridae family (1,(20)(21)(22). By comparison, NPs from −ssRNA viruses with nonsegmented genomes are usually related in both structure and function (1).…”

mentioning

confidence: 99%

Genome encapsidation by orthobunyavirus nucleoproteins

Zheng¹,

Tao²

2013

Proc. Natl. Acad. Sci. U.S.A.

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Structural Basis for RNA Binding and Homo-Oligomer Formation by Influenza B Virus Nucleoprotein

Cited by 58 publications

References 32 publications

The N Terminus of the Influenza B Virus Nucleoprotein Is Essential for Virus Viability, Nuclear Localization, and Optimal Transcription and Replication of the Viral Genome

The N Terminus of the Influenza B Virus Nucleoprotein Is Essential for Virus Viability, Nuclear Localization, and Optimal Transcription and Replication of the Viral Genome

Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

Genome encapsidation by orthobunyavirus nucleoproteins

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