Tacaribe virus (TV), the prototype of the New World group of arenaviruses, comprises a single phylogenetic lineage together with four South American pathogenic producers of hemorrhagic disease. The TV genome consists of two single-stranded RNA segments called S and L. A reconstituted transcription-replication system based on plasmid-supplied TV-like RNAs and TV proteins was established. Plasmid expression was driven by T7 RNA polymerase supplied by a recombinant vaccinia virus. Plasmids were constructed to produce TV S segment analogs containing the negative-sense copy of chloramphenicol acetyltransferase (CAT) flanked at the 5 and 3 termini by sequences corresponding to those of the 5 and 3 noncoding regions of the S genome (minigenome) or the S antigenome (miniantigenome). In cells expressing N and L proteins, input minigenome or miniantigenome produced, respectively, encapsidated miniantigenome or minigenome which in turn produced progeny minigenome or progeny miniantigenome. Both minigenome and miniantigenome in the presence of N and L mediated transcription, which was analyzed as CAT expression. Coexpression of the small RING finger Z (p11) protein was highly inhibitory to both transcription and replication mediated by the minigenome or the miniantigenome. The effect depended on synthesis of Z protein rather than on plasmid or the RNA and was not ascribed to decreased amounts of plasmid-supplied template or proteins (N or L). N and L proteins were sufficient to support full-cycle RNA replication of a plasmid-supplied S genome analog in which CAT replaced the N gene. Replication of this RNA was also inhibited by Z expression.

A fatal case of serologically confirmed hantavirus pulmonary syndrome (HPS) was recently reported in southwestern Argentina. Nucleotide sequence analysis of PCR fragments from conserved regions of the S and M genomic segments of the virus, amplified from RNA extracted from autopsy lung and liver tissues, showed the virus (referred as Andes virus) to be novel. Comparisons between Andes virus genome sequences with the corresponding sequences of the more closely related hantaviruses revealed differences at the amino acid level from 13.6 to 23.9% for G2 protein regions and from 8.5 to 12.5% for the amino terminal region of the nucleocapsid protein. Phylogenetic analysis using the maximum parsimony and maximum likelihood methods showed that Andes virus maps within the clade containing the HPS-associated viruses from North America. Within this group, Andes virus represents a unique lineage. This is, to our knowledge, the first report on the genetic characterization of a hantavirus from South America.

Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. The TV genome encodes four proteins, the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a small RING finger protein (Z). Using a reverse genetic system, we recently demonstrated that TV N and L are sufficient to drive transcription and full-cycle RNA replication mediated by TV-like RNAs and that Z is a powerful inhibitor of these processes (N. López, R. Jácamo, and M. T. Franze-Fernández, J. Virol. 65:12241-12251, 2001). In the present study we investigated whether Z might interact with either of the proteins, N and L, required for RNA synthesis. To that end, we used coimmunoprecipitation with monospecific antibodies against the viral proteins and coimmunoprecipitation with serum against glutathione S-transferase (GST) and binding to glutathioneSepharose beads when Z was expressed as a fusion protein with GST. We demonstrated that Z interacted with L but not with N and that Z inhibitory activity was dependent on its ability to bind to L. We also evaluated the contribution of different Z regions to its binding ability and functional activity. We found that integrity of the RING structure is essential for Z binding to L and for Z inhibitory activity. Mutants with deletions at the N and C termini of Z showed that amino acids within the C-terminal region and immediately adjacent to the RING domain N terminus contribute to efficient Z-L interaction and are required for inhibitory activity. The data presented here provide the first evidence of an interaction between Z and L, suggesting that Z interferes with viral RNA synthesis by direct interaction with L. In addition, coimmunoprecipitation studies revealed a previously unreported interaction between N and L.Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. Within this group the viruses form three phylogenetically distinct clades, one of which includes TV together with the four known South American pathogens that produce severe hemorrhagic disease (Junin, Machupo, Guanarito, and Sabia viruses) (5). TV, however, does not seem to be a human pathogen.TV, like all arenaviruses, is an enveloped virus containing two single-stranded RNA segments called S and L. The S RNA contains two genes, one encoding the nucleoprotein (N; 64 kDa) and one encoding the glycoprotein precursor (55 kDa) (10), while the L RNA encodes the RNA-dependent RNA polymerase (L; 240 kDa) (15) and a small protein with a RING finger motif (Z; 11 kDa) (16). In both S and L RNAs, the genes are arranged in opposite orientation and are separated by noncoding sequences that have the potential to form stable secondary structures (11). The 5Ј regions of arenavirus genomes and antigenomes, though positively stranded, are not translated directly into proteins. Rather, genomes and antigenomes are found only as nucleocapsids tightly bound to N, and the coding sequences are expressed from mRNAs transcribed from the 3Ј regions of the genomes or antigenomes (1,11,17,19,26). These mRNAs contai...

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