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)     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Structural requirements for simian virus 40 replication and virion maturation.

The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.     

Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.

The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.     

Chromosome of the mature virion of simian virus 40 contains H1 histone.

In addition to free SV40 minichromosomes in the compact form, complete virions were obtained from the nuclear extract of productively infected cells. Capsid proteins VP1, VP2, and VP3, as well as histones, were observed on electrophoregrams of proteins prepared from virions. In contrast to the widely accepted view, histone H1 was found in virions in stoichiometric amounts with respect to other histones. The same is true for virions isolated by a conventional method. Free minichromosomes present in infected cells contain all histones and practically no viral proteins.     

Role of simian virus 40 Vp1 cysteines in virion infectivity

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca(2+)-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca(2+) ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.     

Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.     

The transforming proteins of simian virus 40

Simian virus 40 (SV40) is a small DNA tumor virus whose early gene products, large T and small t antigens, efficiently immortalize and transform primary rodent cells, transform rodent cell lines and extend the lifespan of primary human cells. Mutational analysis has revealed that the transforming and lifespan extension properties of large T antigen correlate with binding to and disruption of the normal functions of the human tumor suppressor proteins pRb and p53. Small t antigen contributes to cell proliferation through inactivation of protein phosphatase 2A and subsequent activation of the MAP kinase pathway. By disrupting key cell growth control mechanisms, SV40 transforming proteins provide a valuable system for analysis of cellular growth control mechanisms.     

Replication of the simian virus 40 chromosome with purified proteins.

SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro. Replication started near the origin and proceeded bidirectionally. The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins. When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected. This result indicates that replicated DNA was reconstructed into the nucleosome structure, complexed with parental histones.     

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.     

Selective translation initiation on bicistronic simian virus 40 late mRNA.

We described previously a simian virus 40 (SV40) mutant, pSVAdL, that was defective in synthesis of the late viral protein VP1. This mutant, which contains a 100-base-pair fragment of adenovirus DNA encompassing the major late promoter inserted in the SV40 late promoter region (SV40 nucleotide 294), efficiently synthesizes agnoprotein, a protein encoded by the leader region of the same mRNA that encodes VP1. When the agnoprotein AUG initiation codon in pSVAdL was mutated to UUG, agnoprotein synthesis was abolished, and VP1 synthesis was elevated to wild-type levels. Because levels of late mRNA synthesis were not affected by this mutation, these results support a scanning model of translation initiation and suggest that internal translational reinitiation does not occur efficiently in this situation.     

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.     

Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.