Role of the C Terminus of Lassa Virus L Protein in Viral mRNA Synthesis

Abstract

The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding. L assa virus is a segmented negative-strand RNA virus of the family Arenaviridae causing hemorrhagic fever in humans. The genome consists of two single-stranded RNA segments, each containing two genes in opposite directions (1). The S RNA segment encodes the nucleoprotein (NP) and the glycoprotein precursor. The L RNA encodes the small matrix protein Z (2, 3) and the 200-kDa L protein (4). NP and L protein are the minimal viral trans-acting factors required for replication and transcription of the genome (5-7). L protein mediates the synthesis of two RNA species, mRNA terminating in the intergenic region and noncapped genomic or antigenomic RNA representing a full-length copy of the genome (8, 9). The central domain of L protein harbors the RNA-dependent RNA polymerase (RdRp) (10)(11)(12)(13)(14), and the N terminus contains an endonuclease essential for transcription (15, 16). The latter presumably cleaves off from cellular mRNAs the cap structure with 4 to 5 nucleotides, which serves as a primer for viral mRNA synthesis (9,17,18). Influenza virus, the prototype of "cap-snatching" viruses, expresses a cap-binding protein (PB2) (19) in addition to an endonuclease (PA) (20,21). Assuming the cap-snatching mechanism in arenaviruses corresponds to that in influenza virus, it is tempting to speculate that the L protein also contains a cap-binding domain. We have conducted a mutagenesis study to identify residues that might be involved in cap binding. MATERIALS AND METHODSThe experiments were performed in the context of the T7 RNA polymerase-based Lassa virus replicon system essentially as described previously (5, 14, 16). In brief, L gene mutants were generated by mutagenic PCR using pCITE-L as a template. The PCR products containing the functional cassette for expression of mutant L protein were purified, quantified spectrophotometrically, and used for transfection without prior cloning. As the Phusion polymerase used for PCR has an extremely low error rate (22), more than 95% of the amplified L genes are predictably free of random polymerization errors (23). The presence of the desired artificial mutation was ascertained by sequencing. BSR-T7/5 cells stably expressing T7 RNA polymerase (24) were transfected per well of a 24-well plate with 250 ng of minigenome expressing Renilla luciferase (Ren-Luc), 250 ng of L gene PCR product, 250 ng of pCITE-NP expressing NP, and 10 ng of pCITE-FF-luc expressing firefly luciferase as an internal transfection control. One day after transfection, total RNA was purified for Nort...

Structural insights into reptarenavirus cap-snatching machinery

“…The cap-binding function might have been lost during arenavirus evolution, while the domain might have gained or maintained other functions in virus transcription [13]. A similar situation was proposed for Thogoto virus, an insect transmitted orthomyxovirus.…”

Transcription and replication mechanisms of Bunyaviridae and Arenaviridae L proteins

“…5, see below) of negative strand RNA viruses (NSVs), whereas the N-and C-terminal parts did not exhibit clear protein signatures including RNA helicases or methyltransferases involved in RNA capping (Vieth et al, 2004). Mutation-function studies of the LASV L protein showed that residues critical for function were located both within and outside the predicted polymerase domain and identified specific amino acid residues that were required for RNA transcription, but not replication, mediated by the LASV polymerase in cell-based minigenome (MG) assays (Hass et al, 2008;Lehmann et al, 2014;Lelke et al, 2010). Interestingly, mutations of evolutionary conserved acidic (Asp and Glu) and basic (Lys and Arg) residues within the LASV L protein identified residues that played a critical role in mRNA synthesis without significantly affecting RNA replication .…”

Evolutionary analysis of Old World arenaviruses reveals a major adaptive contribution of the viral polymerase

“…Site numbering refers to Lassa virus ( LASV ) strain AV (Accession ID s: S segment, AF 246121; L segment, AY 179171). In the alignment, few representative OW arenaviruses are shown; conserved sites that affect protein function are in red (Lehmann et al., ), and positively selected sites are highlighted in yellow. SP , Signal Peptide…”

“…Even though the L protein plays a central role in viral genome replication and transcription, structural and functional information is more limited compared to NP. The N‐terminal domain of L has endonuclease activity (Shao, Liang, & Ly, ), which presumably cleaves off the cap structure (Lehmann et al., ). Three of the positively selected sites (S64, E124 and Q125) are located in close spatial proximity to the active site (Figure a).…”