Biochemical characterization of the Lassa virus L protein

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.

Structural and functional characterization of the Severe fever with thrombocytopenia syndrome virus L protein

“…In electrophoretic mobility shift assays, we were able to detect 395 interaction of SFTSV L protein with the conserved termini of all three genomic RNA segments (Figures 2A and B, Supplementary Figure S5). The migration behaviour of the protein-RNA complex in the native gel was different depending on whether 3' or 5' termini were bound (compare Figure 2A consistent with previous findings on the LASV L protein (13). Interestingly, for the S segment, two different sequences of the conserved termini are published which differ from each other by an insertion/deletion of an A at position 9, counted from the 5' end ( Figure 2B).…”

“…As already described for other polymerases (50,51), enzymatic activity was also dependent on divalent metal ions. SFTSV RdRp displayed strong activity in the presence of magnesium ions with an activity plateau reached at Mg 2+ concentrations of >2 mM ( Figure 3C), which is similar to what has been reported for LASV RdRp for manganese ion concentrations (13). The presence of 5 mM Mg 2+ , nucleotides and both 3' and 5' promoter 475…”

“…although the proposed fold of the arenavirus 5' hook has yet to be confirmed by a structure (13,(16)(17)(18).…”

“…Even though further structural insights into distinct functional states of bunyavirus L protein are still lacking, this was a milestone on the path to a complete structural understanding of bunyavirus 65 transcription and replication. In terms of functional data, biochemical characterization of Lassa and Machupo viruses (Arenaviridae family) (13)(14)(15)(16) as well as LACV (12) L proteins have been published. Specific to each bunyavirus family are the 3' and 5' termini of the genomic RNA segments that are highly conserved and almost fully complementary in sequence, denoted as the conserved promoter regions.…”

“…For influenza virus and LACV, the 70 RNA 5' end forms a hook-like secondary structure that is bound in a specific pocket adjacent to the RNA synthesis active site of the L protein (12,17,18). The presence of a hook-conformation and its importance for robust RdRp activity have also been proposed for arenaviruses (13,16). Furthermore, for arenaviruses, it was shown that the L protein initiates genome synthesis de novo by a prime-and-realign mechanism (13) similar to the 75 initiation of influenza virus vRNA synthesis and Hantaan virus genome replication (19)(20)(21)(22).…”