Abstract
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.