Molecular structure and early events in the replication of Tacaribe arenavirus S RNA.

Tacaribe arenavirus S RNA was cloned and analysis of its nucleotide sequence revealed two open reading frames of significant size, one in the virus-sense strand, the other in the virus-complementary strand. The predicted amino acid sequences of the two reading frames were compared with the predicted primary structures of the nucleoprotein (N) and glycoprotein precursor (GPC) of LCM, Pichinde and Lassa viruses. The results indicated a high degree of homology between the proteins of similar properties. It was also found that in Tacaribe virus-infected cells a subgenomic viral-sense GPC RNA and a subgenomic viral-complementary N RNA are synthesized in addition to the full length viral (v) RNA and viral complementary (vc) RNAs. These results support the conclusion that in Tacaribe virus--as in Pichinde and lymphocytic choriomeningitis arenavirus-S RNA encodes the viral N and GPC proteins and has an 'ambisense' coding strategy. Analysis of the S-derived RNA species at early times post-infection in cells incubated with or without inhibitors of protein synthesis indicated that for primary transcription of the N mRNA, protein synthesis is not required; whereas synthesis of the vc RNA, GPC mRNA and v RNA does require protein synthesis to take place.

The 3' end termini of the Tacaribe arenavirus subgenomic RNAs.

Tacaribe virus (TV), a member of the Arenaviridae family, contains two single-stranded RNA genome segments called S and L. Two proteins, in an ambisense coding strategy, are encoded in both the S RNA and the L RNA. The 3' ends of the TV four putative mRNAs have been characterized using S1 nuclease mapping. The experiments revealed that the transcripts terminate within the intergenic region in each RNA segment. No special sequences that might function as termination signals were evident. The 3' end sequences of the four putative mRNAs can be predicted to adopt GC-rich stable hairpin configurations (delta G greater than or equal to -25 kcal). These observations suggest that the transcript structure rather than particular sequences might be the signal involved in the termination of arenavirus transcription.

Tacaribe virus gene expression in cytopathic and non-cytopathic infections.

Tacaribe virus (TV) replication was compared in Vero cells infected under conditions leading either to cell death (c.p.e.(+) infection) or to the establishment of persistence (c.p.e.(-) infection). To this end, two virus preparations were employed: one containing a ratio of standard (plaque-forming) viruses to interfering particles (IP) that would induce a distinct lytic response in Vero cells infected at multiplicities giving synchronous infection and another virus stock enriched in IP that would block the cell-killing potential of the cytolytic virus stock. The following results were obtained: (1) No qualitative differences were observed in the species of intracellular viral RNAs in the lytic infection in comparison with infections leading to persistence or during the early stages of persistence. (2) Levels of viral RNAs were severely reduced when the cells were infected with IP in addition to standard viruses, the RNA accumulation being inversely proportional to the ratio of IP to standard viruses used in the infections. (3) Accumulation of the three measurable mRNAs (those corresponding to the glycoprotein precursor [GPC], to the nucleoprotein [N], and to the p11Z protein) ended earlier in the c.p.e.(-) infections (around 18 hr p.i.) than in the c.p.e.(+) infection (45-68 hr p.i.). (4) The rates of synthesis of the GPC, N, and p11Z proteins were largely determined in both the c.p.e.(+) and c.p.e.(-) infections by the amounts of their corresponding mRNAs. (5) The kinetics of accumulation of the S genomes and also the ratios of the S genome to S antigenome were similar in the different infections (accumulation ending at 45-68 hr p.i.). (6) L genome accumulation proceeded for longer time (until 92 hr p.i.) in the c.p.e.(+) infection than in the c.p.e.(-) infections. In the latter accumulation ended at around 45 hr p.i. Until this time ratios of L genome to L antigenome were similar in the different infections. It is concluded that IP affect virus mRNA synthesis early after infection reducing in this way the rate of viral protein synthesis. Low levels of viral proteins might then limit virus replication. In addition, the results support the idea that in TV infections transcription and replication are independently regulated. The implications of these results with regards to the nature and mode of action of TV IP are discussed.

The 5' region of Tacaribe virus L RNA encodes a protein with a potential metal binding domain.

We have just completed the Tacaribe arenavirus (TV) genome structure by sequencing the 5' region of the L RNA. Analysis of the sequence has indicated the existence of an open reading frame (ORF) in the viral sense RNA encoding a 95 amino acid polypeptide. The first in phase AUG codon is in positions 70-72 from the 5' end of the viral RNA surrounded by a sequence favorable for the initiation of protein synthesis. The ORF ends at positions 355-357. The predicted polypeptide (P11) contains a cysteine-rich sequence bearing a remarkable similarity to the "zinc finger" sequences found in a number of proteins. We have recently reported that the 3' region of the TV L RNA encodes a polypeptide comprising 2210 amino acids in the viral-complementary sequence. This latter gene, i.e., the L gene, terminates at positions 442-440 from the 5' end of the viral RNA. The two genes encoded by the L RNA (L and P11) are in opposite strands of the RNA in sequences that do not overlap, but are separated by a noncoding intergenic region of 82 nucleotides. The nucleotide sequence of the intergenic region leads to the prediction of a strong secondary structure.

Tacaribe virus L gene encodes a protein of 2210 amino acid residues.

The nucleotide sequence of Tacaribe virus (TV) L gene was obtained from two sets of overlapping cDNA clones constructed by walking along the virus L RNA using two successive synthetic DNA primers. Analysis of the sequence indicated the existence of a unique long open reading frame in the viral complementary strand. The first in-phase AUG codon is in positions 31-33 from the 5' end of the viral complementary L RNA surrounded by a sequence favorable for initiation of protein synthesis. The open reading frame ends at positions 6661-6663. The predicted TV L protein is a 2210 amino acid long polypeptide with an estimated molecular weight of 251,942. Comparison of the amino acid sequence of TV L protein with peptide sequences predicted from L-derived cDNA clones of lymphocytic choriomeningitis virus shows an overall 42% of homology.