Purification and properties of RNA polymerase I from Ehrlich ascites cells.

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.

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.

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.

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.

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.

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.

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.

Regulation of ribosomal RNA synthesis in mammalian cells: effect of toyocamycin.

The present study shows that the antitumor agent toyocamycin (4-amino-5-cyano-7beta-D-ribofuranosylpyrrolo(2-3d)pyrimidine) affects rRNA transcription in Ehrlich ascites cells. This action of the antibiotic is dependent on the amino acid composition of the cell culture medium. In cells incubated in a medium rich in amino acids, the high transcription rate of rRNA is lowered by the addition of 2 X 10(-6) M toyocamycin, while in amino acid starved cells the decreased level of rRNA synthesis remains unaffected. Processing of the 45S rRNA precursor is markedly inhibited by toyocamycin in cells incubated in either medium, indicating that the uptake of the drug is unimpaired by amino acid starvation. Toyocamycin does not affect RNA polymerase I (RNA nucleotidyltransferase EC 2.7.7.6) activity when added to in vitro assay systems derived from cells grown in complete or in amino acid deficient media. The drug prevents the activation of rRNA synthesis following the refeeding of amino acid starved cells without affecting the stimulation of protein synthesis.

Regulation of the nucleolar DNA-dependent RNA polymerase by amino acids in Ehrlich ascites tumor cells.

Experiments were performed to ascertain the degree to which the amount of amino acids might be one of the regulatory factors that control the activity of the nucleolar RNA polymerase. Assays of the enzymatic activity were done with isolated nuclei from cells incubated with low and high concentrations of amino acids. Soon after the cells were exposed to a medium enriched in amino acids, a rapid increase of nucleolar RNA polymerase activity occurred. A similar result was obtained in cells incubated with lower concentrations of amino acids. However, the rate of ribosomal RNA synthesized was regularly much higher in cells incubated in a medium enriched with amino acids than in a medium low in amino acids. Apparently, the amino acids only controlled ribosomal RNA synthesis. Thus, neither maturation, processing, and transport of nuclear precursors into cytoplasmic ribosomal RNA, nor the synthesis of rapidly labeled RNA was affected.

Aurintricarboxilic acid as a tool for investigating the template-bound and unbound forms of RNA polymerase I in permeabilized cells.

The presence of template-bound and unbound RNA polymerase I in permeabilized cells was investigated. The two enzyme forms were defined on the basis of their different susceptibilities towards aurintricarboxilic acid (ATA). It was found that addition of ATA to permeabilized cells suppresses initiation of new RNA chains by RNA polymerase I but has no effect on the activity of the enzyme already engaged in transcription. This last activity is not affected even after washing the permeabilized cells for removal of the ATA. The RNA polymerase I activity solubilized from permeabilized cells pre-treated with ATA is 60-70% of that obtained from non-treated controls. The decrease of the solubilized enzyme activity was observed after purification of the enzymes by DEAE-Sephadex columns and cannot be attributed to the presence of inhibitory or activating factors in the enzyme preparations. The simplest interpretation of these findings is that two distinct RNA polymerase I fractions, showing different sensitivities towards ATA are present in permeabilized cells. These fractions should represent the template-bound and unbound RNA polymerase I. The results also show that the amount of ATA-insensitive activity is lower in nuclei than in permeabilized cells, suggesting that detachment of template-bound enzyme occurs during nuclei isolation.

Regulation of rRNA synthesis and processing in animal cells. Effect of nucleoside analogues.

The nucleoside analogues fluorouridine and fluorodeoxyuridine (both at 100 muM) and 8-azaguanine (at 500 muM) inhibit both rRNA transcription and processing in Ehrlich ascites cells. In BHK21 cells fluorodeoxyuridine has no effect on either rRNA maturation or transcription, whereas toyocamycin (at 2 microM) inhibits both processes in BHK21 cells and Ehrlich ascites cells. The drugs inhibit transcription in cells incubated in the complete medium, but have no effect on the decreased transcription in cells incubated in a medium without amino acids. This lack of effect cannot be explained by an altered uptake of the drugs in the amino acid-starved cells, since maturation of the rRNA precursor is affected in cells incubated in media with or without amino acids. The effect of the drugs on rRNA transcription is not the consequence of the inhibition of protein synthesis. The results lend support to the proposal that rRNA processing and transcription are co-ordinately controlled in cells with a high rate of rRNA synthesis.

Increased transcription and decreased degradation control and recovery of liver ribosomes after a period of protein starvation.

In the livers of 5-days-protein-depleted mice there is a decrease of 47% of the ribosome mass. When these animals are fed with an adequate diet, ribosome content is restored to the normal value after 1 day of re-feeding. The mechanisms underlying this phenomenon were studied. It was found that: (1) the activity of RNA polymerase I in the nuclei of livers from re-fed animals showed an enhancement of about 2-fold compared with the activity in normal and protein-depleted liver nuclei; (2) ribosome degradation, measured by the disappearance of radioactivity from ribosomal proteins previously labelled by the administration of NaH14CO3 to the mice, stopped during the first day after re-feeding.

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.

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.

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.

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.