Recombinant Lassa Virus Nucleocapsid Protein (a.a.1-569) [His]

Background: Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV) glycoprotein 1 (GP1), and glycoprotein 2 (GP2); Immunization of non-human primates with viral vectors expressing the arenaviral glycoprotein complex (GPC) confers full protective immunity against a lethal challenge with LASV. Thus, the development of native or quasi native recombinant LASV GP1 and GP2 as soluble, uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever. To this end, mammalian expression systems were engineered for production and purification of secreted forms of soluble LASV GP1 and GP2 proteins. Results: Determinants for mammalian cell expression of secreted uncoupled Lassa virus (LASV) glycoprotein 1 (GP1) and glycoprotein 2 (GP2) were established. Soluble GP1 was generated using either the native glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the intracellular domain (IC) was fused to the ectodomain, and the gene was co-expressed with a complete GP1 gene in cis; (2) the TM and IC domains were deleted and GP1 was co-expressed in cis; (3) expression of GP1 was driven by the native GPC SP. These data implicate GP1 as a chaperone for processing and shuttling GP2 to the cell surface. The soluble forms of GP1 and GP2 generated through these studies were secreted as homogeneously glycosylated proteins that contained high mannose glycans. Furthermore, observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC represents a novel aspect of arenaviral glycoprotein expression. Conclusion: These results implicate GP1 as a chaperone for the correct processing and shuttling of GP2 to the cell surface, and suggest that native GPC SP plays a role in this process. In the absence of GP1 and GPC SP the GP2 protein may be processed by an alternate pathway that produces heterogeneously glycosylated protein, or the polypeptide may not fully mature in the secretory cascade in mammalian cells. The expression constructs developed in these studies resulted in the generation and purification of soluble, uncoupled GP1 and GP2 proteins from mammalian cells with quasinative properties. The observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC establishes new correlates of disease progression and highlights potential opportunities for development of diagnostics targeting the early stages of Lassa fever.

The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro. RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus.