Transcription and RNA replication of tacaribe virus genome and antigenome analogs require N and L proteins: Z protein is an inhibitor of these processes.

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.

Homologous and heterologous glycoproteins induce protection against Junin virus challenge in guinea pigs.

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.

Rapid selection in modified BHK-21 cells of a foot-and-mouth disease virus variant showing alterations in cell tropism.

With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C3Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C3Arg/85 infection. Infection of BHK-Rb cells with C3Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C3-Rb. The selected C3-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9-->Ala in VP3 and either Gly-110-->Arg or His-108-->Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C3Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells.

Genetic characterization and phylogeny of Andes virus and variants from Argentina and Chile.

Andes virus, one of five hantaviruses known to cause hantavirus pulmonary syndrome (HPS), emerged in 1995 in southwestern Argentina (López et al. (1996) Virology 220, 223-226). The complete nucleotide sequence of Andes virus S genome segment was determined and compared with sequences of viral RNAs in autopsy tissues of more recently reported HPS cases from southwestern Argentina and south of Chile (cases ESQ H-1/96 and CH H-1/96). Andes virus S segment was found to be 1876 nucleotides in length and to encode the nucleocapsid protein (N), 428 amino acids in length. S segment analysis also revealed a long 5' non-coding region (547 nucleotides) which displays three copies of an octanucleotide sequence repeat. Comparisons of S segment sequences of ESQ H-1/96 and CH H-1/96 (82% of the entire genome sequence) with the corresponding sequences of Andes virus revealed identities of 97.2% and 98.5%, respectively. Sequence motifs identical and in the same positions as exhibited in Andes virus 5' non-coding region were found in both, ESQ H-1/96 and CH H-1/96 sequences. Three genome fragments of the M segment sequence of the viruses (representing approximately 34% of the entire sequence) were also analyzed. Comparisons of S and M segment sequences of Andes virus with the corresponding sequences of ESQ H-1/96 showed S and M segment identities which differ by less than 1.4%. Andes virus and CH H-1/96 have S segments that differ by 1.5% from one another while their M segment fragments differ by 5.5-8.2%. Phylogenetic analysis showed that Andes virus along with ESQ H-1/96 and CH H-1/96 form a distinct lineage within the clade containing Bayou and Black Creek Canal viruses. It also showed that Andes virus branch of trees derived from comparisons of S or M sequences differed. It is concluded that Andes virus variants causing HPS circulate east and west of the Andes mountains.

Immunological identification of Tacaribe virus proteins.

Tacaribe virus (TV), an arenavirus, is an enveloped virus with genetic information encoded in two segments of single-stranded RNA. The completed sequence of TV led to the identification of four open reading frames (ORF). In order to establish a direct link between ORFs in the sequence of TV and proteins present in virus particles and virus-infected cells, segments of the molecularly cloned TV genome were engineered so as to be expressed in Escherichia coli to produce fusion proteins that were used to raise antisera. The antisera were in turn employed to identify the TV gene products. Serum to the putative nucleocapsid (N) protein reacted with a 68-kDa protein, both in TV particles and in the infected cells. Sera raised to the glycoprotein precursor (GPC) immunoprecipitated two proteins of 68 and 70 kDa from infected cell lysates. Analysis of GPC synthesis in the presence of tunicamycin revealed that the unglycosylated GPC appeared as two polypeptides of 43 and 46 kDa. The putative RNA polymerase gene product (L) was detected as a approximately 240-kDa protein. Serum to the small zinc-binding domain protein (p11-Z) recognized a protein of approximately 11kDa. Immunological evidence is presented that in addition to N and L, two glycoproteins (GP1 and GP2) and p11-Z are structural components of Tacaribe virions.

Genetic identification of a new hantavirus causing severe pulmonary syndrome in Argentina.

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 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 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.

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.

Tacaribe virus infection may induce inhibition of the activity of the host cell Ca2+ and Na+/K+ pumps.

Infection of Vero cells with Tacaribe virus stocks containing a high ratio of standard (plaque-forming) viruses to defective interfering particles (DIP) induced inhibition of the host cell Ca2+ ATPase (Ca2+ pump) and the ouabain-sensitive Na+/K+ ATPase (Na+/K+ pump). The Mg2+ ATPase which is not involved in cation transport was not affected. The presence of DIP in the inocula protected the cells from alteration of the transport-associated ATPases induced by standard viruses.

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.

Transcription of liver ribosomal RNA: differential effect of 5-fluorouracil depending on the nutritional state of mice.

The effect of 5-fluorouracil (5-FUra) on RNA transcription in mice liver cells was studied using animals exposed to different nutritional conditions as a model of normal, nondividing cells. There are two levels of rRNA transcription in mouse liver: a basal level found in mice fed on a complete diet (control mice) and a higher level, two- to three-fold increased over the basal, found in mice fed on a protein-depleting diet for about 3 days and refed on a complete diet for at least 5 hr (refed mice). The rRNA transcription was measured as the activity of RNA polymerase I in isolated liver nuclei. It was found that the intraperitoneal administration of 5-FUra (30 mg/kg body wt) to refed mice rapidly decreases the higher level of rRNA transcription towards the basal level. This is accomplished through a decrease of the initiation frequency of rRNA chains by RNA polymerase I. 5-FUra however, does not affect the basal level of rRNA transcription in liver from mice fed on a complete diet. Under this condition the drug does not affect the initiation frequency of rRNA chains. The effect of 5-FUra on rRNA transcription in refed mice is not mediated by an inhibition of protein synthesis.

Effect of actinomycin D on arenavirus growth and estimation of the generation time for a virus particle.

In an attempt to define the involvement of host transcription in arenavirus growth, a study was made of the effect of actinomycin D (AMD) on the yields of infectious Pichinde, Tacaribe and Junin viruses. The drug was added either immediately after virus adsorption or later after infection, at the stationary phase of virus growth. The time of exposure of the infected cells to the inhibitor was so chosen that the generation and release of virus into the medium took place in the presence of AMD. A double label technique was used to estimate the generation time of an arenavirus particle. This was found to be 6 h or less for all of the arenaviruses examined. The results indicated that treatment of the host cells with AMD, either immediately after virus adsorption or later after infection, does not affect the yield of infectious Pichinde, Tacaribe or Junin viruses, thus implying that continuous host transcription is not required for the replication cycle of these arenaviruses.

Control of rRNA transcription in liver of mice exposed to nutritional changes: initiation frequency may be a regulatory mechanism.

Shortly after feeding protein-depleted mice on a meal containing proteins, the RNA polymerase I activity in isolated liver nuclei shows a two fold to threefold activation over the basal value in nuclei of either normal or protein-depleted mice. This activation can be accounted for by the increase in the number of growing rRNA chains. Moreover, the template-bound RNA polymerase I fraction in nuclei from re-fed mice is about three times that from protein-depleted animals. An excess of template- unbound enzyme was found in liver nuclei from animals under either nutritional condition. Shortly after inhibition of protein synthesis by pactamycin administration to re-fed mice, the number of transcribing RNA polymerase I molecules in liver nuclei decreases to the basal level found in nuclei from protein-depleted mice, while in the latter, protein synthesis inhibition has no effect. These results support the suggestion that short-lived proteins may enhance the initiation frequency by RNA polymerase I after re-feeding.

Effect of tacaribe virus infection on host cell protein and nucleic acid synthesis.

Tacaribe virus stocks were prepared which induced definite lytic responses in Vero cells infected at multiplicities giving synchronous infection. Under these conditions, the first signs of cytopathic effect (c.p.e.) appeared at about 30 h post-infection and cell lysis occurred after 40 h. Before the onset of cytopathic changes, the virus induced inhibition of host cell protein, DNA and RNA (primarily rRNA) synthesis. These were designated c.p.e. (+) virus stocks. The effect of virus on host cell macromolecular synthesis and development of c.p.e. were not related to the virus isolate, but to the conditions under which the virus was produced. Thus, from a single virus clone, working stocks were derived which could or could not induce inhibition of host cell functions and c.p.e. development. The virus stocks that did not induce inhibition are defined as c.p.e. (-). Analysis of [3H]leucine-labelled proteins from Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks revealed synthesis of two virus-specific polypeptides migrating with mobilities corresponding to mol. wt. 68 000 and 79000. These are presumed to correspond, respectively, to the nucleoprotein and to the minor polypeptide p79. In cells infected with the c.p.e. (+) virus stock, the virus-specific polypeptides were synthesized at times when there was a drastic inhibition of host cell protein synthesis. The yield of infectious progeny during the first 24 h of infection is similar in Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks. The proportion of defective interfering particles was much higher in the c.p.e. (-) than in the c.p.e. (+) virus stocks. The results presented here are the first demonstration that an arenavirus affects the biosynthetic machinery of the host cell.

Transcription of ribosomal RNA is differentially controlled in resting and growing BALB/c 3T3 cells.

Shortly after serum-deprived BALB/c 3T3 fibroblasts are stimulated to grow in medium containing 10% calf serum, the RNA polymerase I activity in permeabilized cells shows a two-fold increase over the values observed in either serum-deprived or density-inhibited resting cells. Inhibition of protein synthesis by pactamycin or cycloheximide specifically reduces the enhanced RNA polymerase I activity in serum-stimulated cultures without affecting the values in resting cells. On the other hand, inhibition of rRNA processing by the nucleoside analogs 5-fluoruridine and toyocamycin decreases the rate of 45S rRNA transcription in serum-stimulated cells but has no effect on the values found in resting cultures. These data suggest that the regulation of rRNA transcription occurs by two different mechanisms, depending on the growth state of the cell. One mechanism, in serum-stimulated cells, is dependent on a continuous protein synthesis and a correct 45S rRNA processing; the other, in resting cells, is independent of these two parameters.

Lack of correlation between liver RNA polymerase I and ornithine decarboxylase activities after re-feeding of protein-depleted mice.

When mice fed on a protein-depleted diet are restored to the normal diet (re-feeding), there is a 2-fold increase in liver RNA polymerase I activity. The results obtained with pactamycin; an inhibitor of protein synthesis, suggest the presence of short-lived proteins which are required for inducing an activated state of transcription. To gain an insight on whether ornithine decarboxylase (ODC)--the first enzyme in polyamine biosynthesis--is the labile protein that regulates rRNA synthesis, we have investigated the correlation between liver ODC and RNA polymerase I activities under different nutritional conditions. We have also studied the effects of alpha-difluormethylornithine (alpha-DFMO)--a specific ODC inactivator--on rRNA transcription. The results indicate that, after re-feeding, there is an abrupt increase in ODC activity that rapidly declines, while RNA polymerase I is still increasing. On the other hand, alpha-DFMO--which inhibits the elevated activity of ODC--has not effect on rRNA transcription.

Lack of correlation between liver RNA polymerase I and ornithine decarboxylase activities after re-feeding of protein-depleted mice.

When mice fed on a protein-depleted diet are restored to the normal diet (re-feeding), there is a 2-fold increase in liver RNA polymerase I activity. The results obtained with pactamycin; an inhibitor of protein synthesis, suggest the presence of short-lived proteins which are required for inducing an activated state of transcription. To gain an insight on whether ornithine decarboxylase (ODC)--the first enzyme in polyamine biosynthesis--is the labile protein that regulates rRNA synthesis, we have investigated the correlation between liver ODC and RNA polymerase I activities under different nutritional conditions. We have also studied the effects of alpha-difluormethylornithine (alpha-DFMO)--a specific ODC inactivator--on rRNA transcription. The results indicate that, after re-feeding, there is an abrupt increase in ODC activity that rapidly declines, while RNA polymerase I is still increasing. On the other hand, alpha-DFMO--which inhibits the elevated activity of ODC--has not effect on rRNA transcription.