Electron microscopic demonstration of the presence of amplified sequences at the 5'-ends of the polyoma virus late mRNAs.

Electron microscopic techniques were used to examine the structure of the leader sequences at the 5'-ends of the late polyoma virus mRNAs. The three late mRNA's were partially purified and hybridized to an E. coli plasmid containing two polyoma virus genomes inserted in tandem. The hybrids were spread by the cytochrome c-formamide technique and visualized in the electron microscope. These studies revealed that whereas the body of a given mRNA molecule can hybridize with only one of the two corresponding body sequences in the two adjacent viral genomes, the leader of the same mRNA molecule can hybridize with both copies of the leader sequence-specific DNA. The mVP1 and mVP3 RNA species thus generated hybrids containing two loops, while mVP2 molecules formed hybrids containing one loop. Hence, the leaders of the three polyoma virus late mRNA species must contain two or more repeats of a sequence transcribed from a unique DNA segment. Length measurements showed that most leaders in the late mRNA's consist of at least 200 nucleotides and some contain up to 500 nucleotides, whereas the basic repeat sequence contains about 60 nucleotides.     

Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro.

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.     

Polyoma virus early and late mRNAs in productively infected mouse 3T6 cells.

We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.     

Polyoma Virus DNA: Complete Nucleotide Sequence of the Gene Which Codes for Polyoma Virus Capsid Protein VP1 and Overlaps the VP2/VP3 Genes

The nucleotide sequence of part of the late region of the polyoma virus genome was determined. It contains coding information for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. In the sequence with the same polarity as late mRNA's, all coding frames are blocked by termination codons in a region around 48 units on the physical map. This is the region where the N-terminus of VP1 and the C-termini of VP2 and VP3 have been located (T. Hunter and W. Gibson, J. Virol. 28:240-253, 1978; S. G. Siddell and A. E. Smith, J. Virol. 27:427-431, 1978; Smith et al., Cell 9:481-487, 1976). There are two long uninterrupted coding frames in the late region of polyoma virus DNA. One lies at the 5' end of the sequence and contains potential coding sequences for VP2 and VP3. The other contains 383 consecutive sense codons starting with the ATG at nucleotide position 1,218, extends from 47.5 to 25.8 units counterclockwise on the physical map, and is located where the VP1 gene has been mapped. The VP1 gene overlaps the genes for proteins VP2/VP3 by 32 nucleotides and uses a different coding frame. From the DNA sequence, the amino acid sequence of VP1 was predicted. The proposed VP1 sequence is in good agreement with other data, namely, with the partial N-terminal amino acid sequence and the total amino acid composition. The VP1 coding frame terminates with a TAA codon at 25.8 map units. This is followed by an AATAAA sequence, which may act as a processing signal for the viral late mRNA's. When both nucleotide and amino acid sequences are compared with their counterparts in the related simian virus 40, extensive homologies are found over the entire region of the two viral genomes. Maximum homology appears to occur in those regions which code for the C-termini of the VP1 proteins. The overlap region of VP1 with VP2/VP3 of polyoma virus is shorter by 90 nucleotides than is that of simian virus 40 and shows very limited homology with the simian virus 40 sequence. This leads to the suggestion that the overlap segments of both viruses have been freed from stringency imposed on drifting during evolution and that proteins VP2 and VP3 of polyoma virus may have been truncated by the appearance of a termination codon within the sequence.     

Multiple mRNA species for adenovirus type 2 polypeptides III and pVII.

Fractionation of messenger activities isolated from the cytoplasm of HeLa cells late in infection with adenovirus type 2 reveals that viral polypeptides III and pVII are each synthesized from two different-sized mRNA's. the major messenger activity for each protein has the same sedimentation rate as that previously reported by Anderson et al. (Proc. Natl. Acad. Sci. U.S.A. 71:2756-2760, 1974). The minor messenger activities for III and pVII sediment more rapidly and are not aggregates of the major mRNA's for these proteins. The two minor messenger activities cosediment with two polyadenylated RNA species which are labeled late in infection with 32P and whose molecular weights are estimated to be 2.9 x 10(6) and 2.4 x 10(6). Both of these species hybridize to adenovirus type 2 DNA specific for the mRNA family that is 3' coterminal at adenovirus type 2 map position 49.5 and the mRNA family that is 3' coterminal at 62.0. This is consistent with the possibility that these RNAs have 5'-terminal sequences identical to those of the normal mRNA's for III and pVII but are 3' coterminal at map position 62, the normal 3' terminus of the mRNA's for polypeptides II and pVI. These species are not found in polyadenylated RNA isolated from the nucleus, suggesting that the minor mRNA species are cytoplasmic RNAs.     

Polyoma virus DNA: Sequence from the late region that specifies the leader sequence for late mRNA and codes for VP2, VP3, and the N-terminus of VP1.

The DNA sequence of part of the late region of the polyoma virus genome is presented. This sequence of 1,348 nucleotide pairs encompasses the leader region for late mRNA and the coding sequence for the two minor capsid proteins VP2 and VP3. The coding sequence for the N-terminus of the major capsid protein overlaps the C-terminus of VP2/VP3 by 32 nucleotide pairs. From the DNA sequence the sizes and sequences of VP2 and VP3 could be predicted. Potential splicing signals for the processing of late mRNA's could be identified. Comparisons are made between the sequence of polyoma virus DNA and corresponding regions of simian virus 40 DNA.     

Splice site selection in polyomavirus late pre-mRNA processing.

Polyomavirus late pre-mRNAs contain one 5' splice site and two message body 3' splice sites, which are not used at equal frequencies. As a result of alternative splicing, the total late mRNA population consists of about 5% mVP2 (no message body splice chosen), about 15% mVP3 (promoter-proximal 3' splice site chosen), and about 80% mVP1 (promoter-distal 3' splice site chosen). To determine whether it is splice site strength that determines the ratio of spliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bp surrounding the VP3 3' splice site was substituted with the corresponding region of the VP1 3' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacing between the 3' splice sites. Analysis of RNAs from transfections showed that in each construct, the 3' splice closest to the polyadenylation site was used preferentially. Analysis of a number of additional constructs indicated that there are no strong cis-acting positive or negative regulators of polyomavirus late splicing; rather, splicing choices appear to be determined largely by relative position of splice sites.     

Construction and analysis of viable deletion mutants of polyoma virus.

Viable mutants of polyoma with small deletions ranging in size from 2 to 75 base pairs were obtained by infecting 3T3 cells with polyoma DNA that had been cleaved once with HaeII endonuclease or with DNase-Mn2+ digestion. The HaeII endonuclease-cleaved DNA yielded mutants with deletions at map position 72--73, whereas the mutants generated by DNase I-Mn2+ digestion had deletions either at map position 72--73 or within the map coordinates 92 and 99. Both groups of mutants appeared to grow as well as wild-type virus in 3T3 cells. The deletions at map position 72--73 did not alter the virus's ability to transform rat cells. Hence, the region just to the early side of the origin of DNA replication is not essential for vegetative growth or transformation. But the mutants with deletions in the region between map coordinates 92 and 99, a segment thought to code for polyoma large and middle T antigens (Hutchinson et al., Cell 15:65--77, 1978; Smart and Ito, Cell 15:1427--1437, 1978; Soeda et al., Cell 17:357--370, 1979), transformed rat cells at 0.2 to 0.05 the efficiency of wild-type virus.     

A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs.

Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.     

Characterization of the mRNA's for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.     

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.     

Polyoma virus defective DNAs. II. Physical map of a molecule with rearranged and reiterated sequences (D74).

The structure of the polyoma virus defective species D74 (74% the size of full-length polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24, 24 and 27% in size. The two 24% segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units.Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units are linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin &; Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.