Dissecting the Multifunctional Arenavirus Nucleoprotein

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.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 genomic RNA, and this NP-RNA complex, together with the viral L polymerase, forms the viral ribonucleoprotein (vRNP) complex that directs vRNA replication and gene transcription. Generation of infectious arenavirus progeny requires packaging of these vRNPs into budding particles, a process in which the arenavirus matrix-like protein (Z) plays a central role. In this work, we document the self-association (homotypic interaction) of NP from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), as well as those of the hemorrhagic fever arenaviruses Lassa virus (LASV) and Machupo virus (MACV). We also show heterotypic interaction between NPs from both closely (LCMV and LASV) and distantly (LCMV and MACV) genetically-related arenaviruses. LCMV-NP self-association is dependent on the presence of single-stranded RNA and mediated by the N-terminal region of the NP. We also describe the ability and the domain of LCMV-NP required for the interaction with the Z protein, demonstrating that this domain overlap with a previously documented C-terminal region that counteracts the host type I interferon (IFN-I) response. However, single amino acid mutations affecting the anti-IFN-I function of LCMV-NP did not disrupt the NP-Z interaction, suggesting that within the C-terminal region of NP, different amino acid residues contribute to these two distinct and segregable NP functions. Similarly, we confirm this NP-Z interaction for the HF arenavirus Lassa virus (LASV). Exhaustive analysis in LCMV-NP by the generation of single amino acid mutants demonstrated the important role of the residue D471 in its self-association. Substitutions at this position abrogate NP oligomerization, affecting its ability to replicate and transcribe a Minigenome reporter plasmid. However, its ability to interact with the Z protein, counteract the cellular interferon response and bind to double-stranded (ds)RNA analogs was retained. Additionally, we also document the dominant negative effect of the D471G mutation on viral infection. These results demonstrate that the NP-NP and NP-Z interactions are great potential target for the development of antiviral drugs to combat human-pathogenic arenaviruses