NP and L Proteins of Lymphocytic Choriomeningitis Virus (LCMV) Are Sufficient for Efficient Transcription and Replication of LCMV Genomic RNA Analogs

Abstract

The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5 untranslated region (UTR) and IGR and downstream by the S 3 UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase. Lymphocytic choriomeningitis virus (LCMV) is the prototypic member of the family Arenaviridae, which also includes important human pathogens such as Lassa virus and Junin virus.LCMV provides one of the most widely used model systems to study viral persistence and pathogenesis (40,42). However, the investigation of the molecular mechanisms underlying LCMV persistence and its associated disorders has been hampered by the lack of a genetic system to analyze the structure and function of the LCMV genome and its gene products.The LCMV genome consists of two negative-sense singlestranded RNA segments, designated L and S, with approximate sizes of 7.2 and 3.4 kb, respectively (48, 52). Each RNA segment has an ambisense coding strategy, encoding two proteins in opposite orientation, separated by an intergenic region (IGR) (3,4,56). The S RNA directs synthesis of the three major structural proteins: the nucleoprotein, NP (ca. 63 kDa); and two mature virion glycoproteins, GP-1 (40 to 46 kDa) and GP-2 (35 kDa), that are derived by posttranslational cleavage of a precursor polypeptide, GP-C (75 kDa) (47,55,57). Tetramers of GP-1 and GP-2 make up the spikes on the virion envelope. Evidence indicated that GP-1 mediates virus interaction with host cell surface receptor, which has been recently identified as ␣-dystroglycan (7, 11). The L RNA segment encodes a high-molecular-mass protein (L; ca. 200 kDa) which has the characteristic motifs conserved in all the viral RNAdependent RNA polymerases and a small polypeptide Z (ca. 11 kDa) which contains a RING finger motif and whose function is unknown (26, 50, 52).The NP, the most abundant viral protein in virally infected cells, is associated with the viral RNA (vRNA) to form the nucleocapsid (NC) which is the template for the viral RNA polymerase (26). The L protein is thought to be the main viral comp...

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

“…Arenavirus genomic replication requires L and an appropriate NP-encapsidated RNA template (27,28). Although NP is not essential for the catalytic activity of L itself ( Fig.…”

Differential effect of acute and persistent Junín virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B

“…We propose that at the initial stage of productive infection these proteins might favour virus replication. It was demonstrated that arenavirus N and L proteins are the minimal trans-acting factors necessary to drive transcription and replication of minireplicons (Lee et al, 2000;Ló pez et al, 2001). However, the cellular proteins involved in viral RNA synthesis remain unknown.…”

An interfering activity against lymphocytic choriomeningitis virus replication associated with enhanced mutagenesis

“…LCMV is the prototype arenavirus; its genome includes two RNA segments, L and S, that use an ambisense strategy for gene expression. The L segment encodes the 90 amino acid Z protein, a zinc-finger protein implicated in budding (Pérez et al, 2003;Salvato et al, 1992), and the L protein, the RNA-dependent RNA polymerase, of 2210 amino acids (Fuller-Pace & Southern, 1989;Lee et al, 2000;Lopez et al, 2001;Singh et al, 1987). The S segment encodes the glycoprotein precursor (GP-C) and the nucleoprotein (NP) (Buchmeier et al, 2007).…”