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.     

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.     

Topography of the three late mRNA''s of polyoma virus which encode the virion proteins.

The three cytoplasmic polyadenylated mRNA's which separately encode the three capsid proteins (VP1, VP2, and VP3) of polyoma virus were mapped on the viral genome by one- and two-dimensional gel electrophoreses of nuclease S1-resistant RNA-DNA hybrids. The mRNA's, which we designated mVP1, mVP2, and mVP3 to indicate the coding functions deduced from the cosedimentation of the RNAs and the messenger activities, comprise an overlapping set of 3'-coterminal molecules which also share a heterogeneous family of noncoding 5'-terminal regions (Flavell et al., Cell 16:357--371, 1979; Legon et al., Cell 16:373--388, 1979). The three species differ in the length of the 3' colinear coding region which is spliced to the 5' leader sequences. The common polyadenylated 3' end maps at map unit 25.3. The 5' ends of the colinear bodies of mVP1, mVP3, and mVP2 map at 48.5, 59.5, and 66.5 map units, respectively. An examination of the polyoma virus DNA sequence (Arrand et al., J. Virol. 33:606--618, 1980) in the vicinities of splicing sites approximated by the S1 gel mapping data for sequences common to the ends of known intervening sequences allowed prediction of the precise splice points in polyoma virus late mRNA's. In all three cases, the leader sequences are joined to the mRNA bodies at least 48 nucleotides before the translational initiation codon used in each particular messenger. The start signal which functions in each mRNA is the first AUG (or GUG) triplet after the splice junction.     

Mutation affecting late gene expression in polyoma virus maps in the late region.

A mutation in polyoma virus strain 3049 which results in the overproduction of capsid proteins has been mapped to the late region of the genome between the HindIII site at 45.0 map units and the BamHI site at 58.6 map units. This region contains the coding sequence for VP3 and a portion of VP2, but does not include the late promoters or the coding sequence for the late leaders. The possible role of VP2 or VP3 in the regulation of genetic expression in polyoma virus is discussed.     

The primary sequence of the late region of polyoma virus DNA II. The expression of the late genes and comparison with DNA sequences of SV40 and BKV.

The nucleotide sequence of the late region of the polyoma virus genome has been deduced, which codes for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. The amino acid sequence of VP1 predicted from the nucleotide sequence is in good agreement with the partial N-terminal sequence 1 and amino acid composition of VP1 2,3. When both nucleotide and amono acid sequences are compared with their counterparts in the related viruses, SV40 4,5 and BKV (R. Young, personal communication), extensive homologies are found along the entire regions of the viral genes. Maximum homologies appear to occur in the regions which code for the C-terminal of VP1, on the contrary of the result of heteroduplex analysis 6 with 6 with SV40 and polyoma virus DNAs.     

Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40.

The nucleotide sequence of the late region of the polyoma genome has been determined. It consists of 2366 bp and encodes the virion capsid proteins VP1, VP2 and VP3. Extensive open reading frames identify the possible coding sequences of VP2 and VP3 toward the 5′ end of the late region, and of the major capsid protein VP1 toward the 3′ end of the late region. The 5′ end of the sequence encoding VP1 overlaps the 3′ VP2/VP3 region by 29 nucleotides and is in a different reading frame. The predicted amino acid sequences for all three known capsid proteins show extensive homology with the analogous capsid proteins of SV40 throughout most of their length. The VP2/VP3 amino acid homology between the two viruses is 34%, while the major capsid protein VP1 is much more highly conserved, showing 54% homology. These homologies together with the extent of open reading frames help to define the extent of the coding sequences. The VP2 initiator begins at position 269 and the coding region extends to the first termination codon beginning at 1226. The predicted size of VP2 is 35,007 daltons. A probable VP3 initiator is within the VP2 coding sequence at position 614 and is in the same frame as VP2. This coding sequence can also utilize the terminator at position 1226, and the predicted size of the VP3 translation product is 22,979 daltons. The VP1 coding region begins at position 1197 and continues in a frame different from that of VP2/ VP3 to a termination point at 2349. The molecular weight of VP1 is predicted to be 42,834 daltons. The 5′ untranslated region contains sequences that resemble a potential ribosomal binding site and a possible mRNA capping sequence similar to those found in other eucaryotic systems. There is also a sequence (5′-TCAAGTAAGTGA-3′) almost identical to one found in two regions containing potential splice sites in the early region of polyoma. The 5′ untranslated region does not show the extensive repeated sequences found in the similar region of SV40. The 3′ untranslated region contains the sequence 5′-AATAAA-3′, thought to represent a polyadenylation signal. As in the early region of polyoma, the extensive nucleotide and deduced amino acid homology with SV40 indicate a close evolutionary relationship between the two viruses, and help to identify regions of common and important structure-function relationships.     

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.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.     

Nucleotide sequence of the restriction fragment Hind-F-EcoRI1 of simian-virus-40 DNA (part of the VP1 gene).

The nucleotide sequence of the simian virus 40 (SV40) genome region between the cleavage sites for restriction endonucleases EcoRI (map position 0) and HindII (map position 0.05) has been determined mainly by the partial chemical DNA degradation procedure of Maxam and Gilbert. This fragment represents 5.3% of the genome of SV40 and is located in the late region, internally in the VP1 gene. The message strand shows only one open reading frame for translation into protein, which connects to the one for the preceding fragment. On this basis part of the amino acid sequence of the VP1 protein is presented.     

Polyoma viruses with mutations at endonuclease HindII site 1: alterations at the COOH terminus of VP1.

Four mutants of polyoma virus lacking endonuclease HindII site 1 were isolated and characterized with respect to the VP1 coding sequence. Three of these mutants had deletions that removed 0.2 to 0.3% of the genome. All three deletion mutants encoded VP1 proteins that were smaller than wild type and that lacked one or more tryptic peptides normally found in the wild-type VP1 protein. Our results suggest the HindII site 1 is at, or very near, the carboxy terminal end of the coding sequence for VP1. A model for the peptide organization in that region is presented.     

Polyoma virus has three late mRNA''s: one for each virion protein.

Polyoma virus mRNA, isolated from the cytoplasm of 3T6 cells late after infection and purified by hybridization to HpaII fragment 3 of polyoma virus DNA, was separated on 50% formamide-containing sucrose density gradients, and the fractionated RNA was recovered and translated in vitro. Analysis of the cell-free products showed that the minor virion protein VP3 was synthesized from an mRNA sedimenting at approximately 18S betweeen the 19S VP2 mRN and the 16S VP1 mRNA. Other experiments showed that the VP2 and VP3 can be labeled with formyl methionine from initiator tRNA. We conclude that there are three late polyoma virus mRNA's, each directing the synthesis of only one viral capsid protein.     

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.     

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.     

Human polyomavirus JC virus genome.

The complete DNA sequence of the human JC virus, which was found to consist of 5,130 nucleotide pairs, is presented. The amino acid sequence of six proteins could be deduced: the early, nonstructural proteins, large T and small t antigens; the late capsid proteins, VP1, VP2, and VP3; and the agnogene product encoded within the late leader sequence, called the agnoprotein in simian virus 40. The extent of homology between JC virus DNA and the genomes of simian virus 40 (69%) and BK virus (75%) confirmed the close evolutionary relationship of these three polyomaviruses. The sequences showing the greatest divergence in these viral DNAs occurred within the tandem repeats located to the late side of the replication origins.     

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. The early region and its T-antigens.

The DNA sequence of the early coding region of polyoma virus is presented. It consists of 2739 nucleotides. The sequence predicts that more than one reading frame can be used to code for the three known polyoma virus early proteins (designated small, middle and large T-antigens). From the DNA sequence, the 'splicing' signals used in the processing of viral RNA to functional messenger RNAs can be predicted, as well as the sizes and sequences of the three proteins. Other unusual aspects of the DNA sequence are noted. Comparisons are made between the DNA sequences and the predicted amino acid sequences of the respective large T-antigens of polyoma virus and the related virus Simian Virus (SV) 40.     

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.     

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).