Translational control of synthesis of simian virus 40 late proteins from polycistronic 19S late mRNA.

The simian virus 40 (SV40) 19S late mRNA is polycistronic, encoding multiple late proteins: agnoprotein, VP2, and VP3. We constructed a chloramphenicol acetyltransferase (CAT) transient expression vector in which the SV40 sequences between nucleotides 5171 and 1046 (via the SV40 origin of replication and including the late promoter) were inserted 5' to the cat gene; therefore, the AUG for CAT expression occurs after the AUGs for agnoprotein, VP2, and VP3. CAT enzyme activity assayed after transfection of these constructions indicates the level of CAT AUG utilization and, therefore, can be used as a measure of the ability of prior AUGs to intercept scanning ribosomes. Specifically, deletions and point mutations of the viral AUGs resulted in increased CAT enzyme activity owing to increased utilization of the downstream CAT AUG. To compare a variety of mutants, we used the levels of increase to calculate the translational efficiency of the viral AUGs. Some of our data agree with predictions of the modified scanning model (MSM). Little variation in downstream CAT AUG utilization was noted regardless of whether the VP2 AUG (in a weak MSM sequence context) was intact or removed. Hence, a scanning ribosome may easily bypass it. Similar analysis of the VP3 AUG (in a favorable MSM sequence context) demonstrated that it could efficiently intercept ribosomes prior to the downstream AUG. Overall, these data indicate that the structure of the 19S late mRNA and the relative efficiency of translational start codon utilization can account for the VP3/VP2 ratio found in infected cells. The agnoprotein reading frame, depending on how the mRNA precursor is spliced, is either not contained in the mRNA or is terminated near the VP2 AUG. Under these conditions, the ability of the agnoprotein AUG to block downstream CAT AUG utilization was found to be minimal in our assay. However, we directly tested the blocking ability of the agnoprotein AUG under conditions in which the reading frame terminated well after the CAT AUG. Although the agnoprotein AUG lies in a very good sequence context, this direct analysis showed that it interfered minimally with utilization of the CAT AUG when under the control of the SV40 late promoter. However, expected high levels of interference were regained when the late promoter was replaced with the Rous sarcoma virus long terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leakage of nuclear transcripts late in simian virus 40-infected CV1 cells: quantitation of spliced and unspliced late 19S RNAs.

During the late phase of simian virus 40 infections of CV1 cells, the relative ratios of the spliced to the unspliced RNA molecules of the 19S family were measured. In the cytoplasm, unspliced 19S RNA represented between 1 and 2% of spliced 19S RNA. This ratio could be altered by the use of different methods of RNA extraction such that unspliced RNA was observed at 10 to 20% of spliced RNA. The nuclear ratios of spliced to unspliced 19S RNA were also determined. In contrast to cytoplasmic RNA, nuclear unspliced RNA was several hundred percent that of nuclear spliced 19S RNA. Cytoplasmic unspliced 19S RNA appears to be of nuclear origin, and its presence in the cytoplasmic fraction is due to nuclear leakage during RNA fractionation.     

Nucleotides sequence of the genes for the simian virus 40 proteins VP2 and VP3.

We have determined the nucleotide sequence of the DNA of simian virus 40. The proceeding report (Dhar, R., Reddy, V.B., and Weissman, S.M. (1978) J. Biol. Chem. 253, 612-620) presents the sequence of a portion of the simian virus 40 DNA that overlaps the region encoding the 5' end of the minor structural protein VP2. We report here the sequence of the remainder of the genes for minor structural proteins VP2 and VP3. The results indicate that the mRNA for the two proteins is read in the same phase and the initiation site for VP3 lies within the structural gene of VP2. The codons of the COOH-terminal amino acids of VP2 and VP3 are read in a second phase as the codons of the NH2-terminal amino acids of VP1.     

Capping structures of simian virus 40 19S and 16S mRNAs.

In vivo [methyl 3H]-labeled SV40 19S and 16S mRNA species were purified and their internal methylation as well as their capping structures analyzed. SV40 viral mRNA sedimenting in the 19S region contains approximately equal proportions of m7GpppAm and m7Gppm6Am, while the 16S mRNA contains mainly m7Gpppm6Am. N6 methyl adenosine is located internally within the RNA chains of both the 19S and 16S species.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.     

The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.

A cDNA fragment coding for poliovirus capsid polypeptide VP1 was inserted into a simian virus 40 (SV40) genome in the place of the SV40 VP1 gene and fused in phase to the 3' end of the VP2-VP3 genes. Simian cells were infected with the resulting hybrid virus in the presence of an early SV40 mutant used as a helper. Indirect immunofluorescence analysis of the infected cells using anti-poliovirus VP1 immune serum revealed that the SV40/poliovirus fusion protein was located inside the cell nucleus. Deletions of various lengths were generated in the SV40 VP2-VP3 portion of the hybrid gene using BAL31 nuclease. The resulting virus genomes expressed spliced fusion proteins whose intracellular location was either intranuclear or intracytoplasmic, depending on the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed by site-directed mutagenesis of the Lys residue at position 320. Modification of Lys-320 into either Thr or Asn abolished the nuclear accumulation of the fusion protein. It is concluded that at least part of the sequence of VP2 amino acids 317 to 323 allows VP2 and VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cell cytoplasm to the cell nucleus by interaction, with VP1 acting as a carrier.     

Isolation of a monoclonal antibody viral polypeptide VP2 of simian virus 40

A stable hybridoma cell line, IIG8-203-2, that secretes a monoclonal antibody of the immunoglobulin subclass M was obtained by fusion of mouse myeloma cells with spleen cells of mice that had been immunized with the viral polypeptide VP2 of simian virus 40. The monoclonal antibody recognizes viral polypeptides that migrate with VP2 polypeptides in a sodium dodecyl sulfate-polyacrylamide gel. It also recognizes two intracellular polypeptides (29,000 and 37,000 daltons) in a detergent-insoluble fraction extracted 30 h after virus infection of TC7 cells.