Translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' cap.

The major late 16S mRNA species of simian virus 40 encodes both a 61-amino-acid protein, LP1, and the major virion protein, VP1. Although the initiation signal GCCAUGG is usually utilized at high efficiency, at least one-third of 40S ribosomal subunits bypass it when it is present on the major 16S mRNA of simian virus 40 (S. A. Sedman, P. J. Good, and J. E. Mertz, J. Virol. 63:3884-3893, 1989). The LP1 translation initiation codon is situated 10 bases from the 5' end of this mRNA. To determine whether the short length of the untranslated leader of this mRNA affects the efficiency of translation initiation at the LP1 initiation signal, monkey cells were transfected with plasmids which encode major late 16S-like mRNAs with 5' untranslated regions (UTRs) of 6 or 44 bases. Decreasing the length of the 5' UTR from 44 to 6 bases resulted in a 30% decrease in translation initiation at the LP1 AUG and a threefold increase in synthesis of VP1. When the VP1 open reading frame was replaced with the chloramphenicol acetyltransferase open reading frame, the reduction in 5' UTR length resulted in a 70% decrease in translation initiation at the LP1 AUG and a 30% increase in chloramphenicol acetyltransferase synthesis. Therefore, ribosomes bypass an AUG codon more efficiently when it is located very close to the 5' end of the mRNA.     

Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40.

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.     

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.     

Ability of nonpermissive mouse cells to express a simian virus 40 late function(s)

Mouse cells are fully nonpermissive for simian virus 40 (SV40). Infection does not lead to detectable virus replication. In this report, it was shown, first, that spliced 16S and 19S SV40 late mRNA were present in cytoplasmic and polysomal polyadenylated acid+ RNA preparations from SV40-infected baby mouse kidney cells. The 16S and 19S SV40 late mRNA's produced in infected baby mouse kidney cells were identical to or similar to the 16S and 19S SV40 late mRNA's produced in permissive monkey cells as judged by their S1 mapping patterns performed with the late strand of HpaII-BamHI fragment B and by their sedimentation patterns in a sucrose gradient. It was also shown that the 16S late mRNA from infected baby mouse kidney cells could be translated into a polypeptide which was identical to or similar to virion protein VP1 in every aspect examined, including the patter of peptide mapping by limited proteolysis. Second, we reported that mouse kidney cells produced detectable, although low, levels of SV40 virion protein VP1, as shown by the sodium dodecyl sulfate-polyacrylamide gel autoradiogram of [35S]methionine-labeled proteins immunoprecipitated by a rabbit antiserum directed against SV40 virion proteins. Third, it was reported that infected baby mouse kidney cells produced late mRNA's either (i) when the infection was done at a restrictive temperature with the nonleaky tsA58 mutant or (ii) in cells treated with 100 microgram of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. This suggested that large T antigen was not required for the synthesis of late mRNA in mouse cells.     

Mechanisms of synthesis of virion proteins from the functionally bigenic late mRNAs of simian virus 40.

The late 19S RNAs of simian virus 40 (SV40) are functionally polycistronic, i.e., all encode both VP2 and VP3. The VP3-coding sequences are situated in the same reading frame as the VP2-coding sequences, within the carboxy-terminal two-thirds of the VP2-coding sequences. To test whether VP3 is produced by proteolytic processing of VP2, we introduced a variety of deletion and insertion mutations within the amino-terminal end of the VP2-coding sequences. Genetic and biochemical analysis of the proteins synthesized in cells transfected with these mutants indicated that VP2 and VP3 were synthesized independently of each other. A leaky scanning model for the synthesis of VP3 was tested by the insertion of a strong initiation signal (CCAACATGG) upstream of the VP3-coding sequences. When the signal was placed in the same reading frame as VP3, synthesis of VP3 was reduced by a factor of 10 to 20, whereas synthesis of the expected VP3-related fusion protein occurred at a rate similar to that observed for VP3 in cells transfected with wild-type SV40 DNA. Insertion of this strong initiation signal at the same site, but in a different reading frame, resulted in the synthesis of VP3 at one-third of the wild-type rate. Mutation of the VP2 initiator AUG resulted in a small but reproducible (1.6-fold) increase in VP3 accumulation. From these experiments we conclude that (i) VP3 is synthesized predominantly by independent initiation of translation via a leaky scanning mechanism, rather than by proteolytic processing of VP2 or direct internal initiation of translation; (ii) a strong initiation signal 5' of the VP3-coding sequences can significantly inhibit synthesis of VP3, but does not act as an absolute barrier to scanning ribosomes; (iii) approximately 70% of scanning ribosomes bypass the VP2 initiator AUG, which is present in a weak context (GGUCCAUGG), and initiate at the VP3 initiation signal located downstream; and (iv) reinitiation of translation appears to occur on the SV40 late 19S mRNAs at an efficiency of 25 to 50%.     

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader.

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.     

Leaky scanning and scanning-independent ribosome migration on the tricistronic S1 mRNA of avian reovirus

The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.     

Simian virus 40 maturation in cells harboring mutants deleted in the agnogene

The predominant leader region of the late 16 S mRNAs of simian virus 40 encodes a histone-like, 61-amino acid, DNA-binding protein called the agnoprotein or LP1. To test the hypothesis that this protein facilitates assembly of viral minichromosomes into virions, we have studied the synthesis of virions in cells infected with mutants deleted in this region of the SV40 genome. We found that 220 S mature virions, indistinguishable from those of wild type, were produced in cells infected with these mutants. As in wild-type-infected cells, no assembly intermediates other than 75 S chromatin were observed. However, data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced virions more slowly than cells infected with wild-type virus. Taken together with data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, these findings strongly suggest that LP1 plays a role in expediting virion assembly.     

Missense mutations in the VP1 gene of simian virus 40 that compensate for defects caused by deletions in the viral agnogene.

Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smaller plaques than does wild-type virus. Within three passages at low multiplicities of infection, virus stocks of several such mutants accumulated variants that synthesized an altered form of the major virion protein, VP1, having a slightly faster mobility in sodium dodecyl sulfate-polyacrylamide gels than did the wild-type protein. Because these variants overgrew the original virus stocks, we consider them to be second-site revertants. By construction and characterization of a series of recombinants, the second-site mutations were shown to map to at least two different regions of the VP1 gene. Nucleotide sequence analysis indicated that single-amino-acid changes were responsible for the rapid mobility of VP1. When combined in cis with either a wild-type or mutant leader region, these VP1 mutations sped up by 10 to 20 h the time course of accumulation of infectious progeny but not of viral DNA or VP1. LP1, the protein encoded by the agnogene, was shown previously to be necessary for the efficient transport of the virion proteins to the nucleus or for their efficient assembly with viral minichromosomes. The VP1 missense mutations reported here compensate for the lack of LP1 by facilitating this process. On the basis of these findings and findings reported previously by us and others, we hypothesize that LP1 facilitates the formation of infectious particles by inhibiting the polymerization of VP1 molecules until the time they interact with viral minichromosomes; the VP1 mutations reported here compensate for the loss of LP1 by lessening the potential of VP1 for self-polymerization.     

Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome.

The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP3 corresponds to the carbosyl terminal part of VP2, and considering various evidence which indicates that the SV40 Hind E segment is part of the amino acid sequence of VP2-VP3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP1 (Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the vp3 poly peptide, and the amino acid composition is in good agreement with published values (Greenaway and Levine, 1973).     

Varicella-zoster virus open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters.

The varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein is the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. These are two virion tegument proteins that have extensive amino acid sequence identity in their amino-terminal and middle domains. ORF10, however, lacks the acidic carboxy terminus which is critical for transactivation by VP16. Earlier studies showed that VZV ORF10 does not form a tertiary complex with the TAATGARAT regulatory element (where R is a purine) with which HSV-1 VP16 interacts, suggesting that ORF10 may not have transactivating ability. Using transient-expression assays, we show that VZV ORF10 is able to transactivate VZV immediate-early (IE) gene (ORF62) and HSV-1 IE gene (ICP4 and ICP0) promoters. Furthermore, cell lines stably expressing ORF10 complement the HSV-1 mutant in1814, which lacks the transactivating function of VP16, and enhance the de novo synthesis of infectious virus following transfection of HSV-1 virion DNA. These results indicate that ORF10, like its HSV-1 homolog VP16, is a transactivating protein despite the absence of sequences similar to the VP16 carboxy-terminal domain. The transactivating function of the VZV ORF10 tegument protein may be critical for efficient initiation of viral infection.     

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production.     

Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.