The C-terminal region of lymphocytic choriomeningitis virus nucleoprotein contains distinct and segregable functional domains involved in NP-Z interaction and counteraction of the type I interferon response.

Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Journal of Virology (Impact Factor: 4.65). 12/2011; 85(24):13038-48. DOI: 10.1128/JVI.05834-11
Source: PubMed

ABSTRACT Several arenaviruses cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. Arenavirus nucleoprotein (NP), the most abundant viral protein in infected cells and virions, encapsidates the viral genome RNA, and this NP-RNA complex, together with the viral L polymerase, forms the viral ribonucleoprotein (vRNP) that directs viral RNA replication and gene transcription. Formation of infectious arenavirus progeny requires packaging of vRNPs into budding particles, a process in which arenavirus matrix-like protein (Z) plays a central role. In the present study, we have characterized the NP-Z interaction for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). The LCMV NP domain that interacted with Z overlapped with a previously documented C-terminal domain that counteracts the host type I interferon (IFN) response. However, we found that single amino acid mutations that affect the anti-IFN function of LCMV NP did not disrupt the NP-Z interaction, suggesting that within the C-terminal region of NP different amino acid residues critically contribute to these two distinct and segregable NP functions. A similar NP-Z interaction was confirmed for the HF arenavirus Lassa virus (LASV). Notably, LCMV NP interacted similarly with both LCMV Z and LASV Z, while LASV NP interacted only with LASV Z. Our results also suggest the presence of a conserved protein domain within NP but with specific amino acid residues playing key roles in determining the specificity of NP-Z interaction that may influence the viability of reassortant arenaviruses. In addition, this NP-Z interaction represents a potential target for the development of antiviral drugs to combat human-pathogenic arenaviruses.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Influenza A and B viruses co-circulate in humans, and together cause disease and seasonal epidemics. These two types of influenza virus are evolutionarily divergent, and exchange of genetic segments inside co-infected cells occurs frequently within types, but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that full-length influenza hemagglutinin (HA) of prototype B viruses can complement the function of multiple influenza A viruses. We show that viral non-coding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B HA segment can not be incorporated into influenza A virions. However, by adding the influenza A packaging signals to full length influenza B glycoproteins, we rescued influenza A viruses that possessed HA, NA or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be at least in part attributed to incompatibilities in the viral-specific packaging signals required for effective segment incorporation into nascent virions.
    Journal of Virology 07/2014; 88(18). DOI:10.1128/JVI.01440-14 · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The X-ray crystal structure of the Lymphocytic choriomeningitis virus nucleoprotein C-terminal immunosuppressive domain (LCMV NPΔ340) was determined to 2.0 Å resolution. The structure indicates that LCMV NPΔ340, like the other structurally characterized arenaviral nucleoproteins, adopts the fold of an exonuclease. This structure provides a crucial three-dimensional template for functional exploration of the replication and immunosuppression of this prototypic arenavirus.
    Acta Crystallographica Section D Biological Crystallography 06/2014; 70(Pt 6):1764-1769. DOI:10.1107/S1399004714007883 · 7.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of Virology 04/2014; 88(11). DOI:10.1128/JVI.00321-14 · 4.65 Impact Factor