Characterization of the Genomic Promoter of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

Abstract

The genome of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense, single-strand RNA segments designated L and S. Arenavirus genomes exhibit high sequence conservation at their 3 ends. All arenavirus genomes examined to date have a conserved terminal sequence element (3-terminal 20 nucleotides [nt]) thought to be a highly conserved viral promoter. Terminal complementarity between the 5 and 3 ends of the L and S RNAs predicts the formation of a thermodynamically stable panhandle structure that could contribute to the control of RNA synthesis. We investigated these issues by using a transcription-and replication-competent minireplicon system. A series of overlapping deletions spanning the 3-terminal 20-nt region of an LCMV minigenome (MG) was generated, and the mutant MGs were analyzed for their activity as templates for RNA synthesis by the LCMV polymerase. The minimal LCMV genomic promoter was found to be contained within the 3-terminal 19 nt. Substitution of C for G at the last 3-end nucleotide position in the MG resulted in nondetection of RNA transcription or replication, whereas the addition of a C at the 3 end did not have any significant affect on RNA synthesis mediated by the LCMV polymerase. All other mutations introduced within the 3-terminal 19 nt of the MG resulted in undetectable levels of promoter activity. Deletions and nucleotide substitutions within the MG 5 end that disrupted terminal complementarity abolished chloramphenicol acetyltransferase expression and RNA synthesis mediated by the LCMV polymerase. These findings indicate that both sequence specificity within the 3-terminal 19 nt and the integrity of the predicted panhandle structure appear to be required for efficient RNA synthesis mediated by the LCMV polymerase.The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is one of the most widely used model systems to study virus-host interactions, such as viral persistence and associated disease (8, 39). The LCMV genome is composed of two negative-sense single-stranded RNA segments, called S (3.2 kb) and L (7.2 kb) (46, 49). Both segments use an ambisense coding strategy to direct synthesis of two proteins from two open reading frames with opposite orientation and separated by an intergenic region (IGR) (2, 3, 60). The S RNA encodes the nucleoprotein NP (ca. 63 kDa) and the glycoprotein precursor GP-C (75 kDa). GP-C is posttranslationally cleaved to yield the mature glycoproteins GP-1 (40 to 46 kDa) and GP-2 (35 kDa) (45,54,62). Tetramers of GP-1 and GP-2 form the spikes on the virion envelope and mediate virus interaction with the cellular receptor (9, 11). The L RNA encodes the virus RNA-dependent RNA polymerase (RdRp) (L, ca. 200 kDa) (21, 30, 51) and a small (11-kDa) RING finger protein (Z) (49). NP and L are associated with the viral RNA to form ribonucleoprotein (RNP) complexes, which are active in transcription and replication (14,22). As with other negative-strand RNA viruses, this RNP is the minimal infectious unit.All cis-...

The small RING finger protein Z drives arenavirus budding: Implications for antiviral strategies

“…All plasmid DNA was prepared by using Qiagen (Valencia, CA) technology. At 72 h posttransfection, supernatants (SP) (1.5 ml) were saved, and CE were prepared for CAT assay as described (29). Aliquots of the clarified SP (600 l) were used to infect fresh monolayers of BHK-21 cells in 12-well plates.…”

“…Plasmid pMG#7⌬2G was generated by deleting the two 3Ј termini G residues of the T7RP promoter in plasmid pMG#7 (29). In cells expressing T7RP, pMG#7⌬2G allows for synthesis of a minigenome (MG) RNA consisting of the 5Ј and 3Ј UTR and intergenic region of the LCMV ARM S segment, and a chloramphenicol acetyltransferase (CAT) reporter gene substituting for the NP ORF.…”

Genome comparison of virulent and avirulent strains of the Pichinde arenavirus

“…The genes are encoded in an ambisense orientation [7], such that the NP and L genes are encoded in the conventional negative sense, whereas the GPC and Z genes are encoded in the positive sense. At both ends of the RNA segments, the terminal 19 nucleotides, conserved among all arenaviruses, are complementary to each other and are predicted to form panhandle structures that are the cis-acting signals required for viral RNA transcription and replication [21,40]. A unique feature among the arenavirus genomic RNAs is the noncoding intergenic regions (IGRs) located between the two ORFs on each of the segments [7].…”

Efficient Rescue of Recombinant Lassa Virus Reveals the Influence of S Segment Noncoding Regions on Virus Replication and Virulence

“…arenavirus, LCMV, the 5Ј-and 3Ј-terminal 19 nucleotides (nt) were found to be essential for maintaining promoter function, as mutation of these residues (nt 1 to 19) severely reduced or abolished promoter activity (24). However, deletions internal to the predicted panhandle structure (positions 20 to 23 or 24 to 33) did not reduce LCMV promoter activity.…”

“…The composition of the minimum promoter element in the S segment of LCMV and LASV has been studied previously (14,24), and a critical role for the terminal 19 nt has been shown. An analysis of the predicted secondary structure of the LASV S segment RNA (Mfold [29]) revealed this well-described 19-nt terminal panhandle, but interestingly, a second high-energy panhandle structure could involve base pairing up to position 33 ( Fig.…”