The sequences between 0.59 and 0.54 map units on SV40 DNA code for the unique region of small t antigen.

To demonstrate directly that the carboxy terminal portion of simian virus 40 (SV40) small t is encoded by a sequence of nucleotides from the region between 0.59-0.54 map units on SV40 DNA, we characterized the putative shortened forms or fragments of small t produced by mutants of SV40 (dl 884, dl 885, dl 890) with deletions in this region of the genome. Attempts to isolate the putative fragments of small t from mutant-infected cells, or from cell-free systems primed with mRNA from mutant-infected cells, resulted in only low yields of the fragments. Experiments using purified SV40 mRNA in low background cell-free systems, in which large T and small t could be detected without immunoprecipitation, suggested that these low yields were accounted for by reduced amounts of mRNA coding for the shortened forms of small t present in the mutant-infected cells. Larger amounts of putative fragments of small t were produced by translation of deletion mutant cRNA (complementary RNA synthesized in vitro using purified deletion mutant DNA and E. coli RNA polymerase). Fingerprint analysis of the proteins produced showed that they contain most, if not all, of the methionine peptides common to small t and large T. Furthermore, the fragments of small t produced in response to dl 884 and dl 890 lack two methionine peptides that are present in small t but not in large T. These data provide direct evidence that the region between 0.59-0.54 map units on SV40 DNA codes for polypeptide sequences that are unique to small t, and establishes that the nucleotide sequences from the region between 0.59-0.54 map units are both a coding sequence (for small t) and an intervening sequence (for large T).     

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.     

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.     

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.     

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.     

Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.     

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.     

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.     

Nuclear localization of poliovirus capsid polypeptide VP1 expressed as a fusion protein with SV40-VP1

The poliovirus cDNA fragment coding for capsid polypeptide VP1 was inserted between the EcoRI and BamHI sites of SV40 DNA, generating a chimaeric gene in which the sequence of the 302 amino acids (aa) of poliovirus capsid polypeptide VP1 was placed downstream from that of the 94 N-terminal aa of SV40 capsid polypeptide VP1. The resulting defective, hybrid virus, SV40-delta 1 polio, was propagated in CV1 cells using an early SV40 mutant, am404, as a helper. Cells doubly infected by SV40-delta 1 polio and am404 expressed a 50-kDal fusion protein which was specifically immunoprecipitated by polyclonal and/or monoclonal antibodies raised against poliovirus capsids or against poliovirus polypeptide VP1. Examination of the infected cells by immunofluorescence after staining with anti-poliovirus VP1 immune sera revealed that the fusion protein was mostly located in the intra- and perinuclear space of the cells, in contrast to the exclusively intracytoplasmic location of genuine poliovirus VP1 polypeptide that was observed in poliovirus-infected cells. This suggests that the N-terminal part of the SV40-VP1 polypeptide could contain an important sequence element acting as a migration signal for the transport of proteins from the cytoplasm to the nucleus.     

The sequence of the 3'' non-coding region of the hexon mRNA discloses a novel adenovirus gene.

We report the sequence of a 1164 nucleotide long DNA segment, located between map positions 59.5 and 62.8 on the adenovirus type 2 genome. The sequence comprises the 701 nucleotides long 3' non-coding region of the hexon mRNA as well as several important processing signals. The sequence revealed unexpectedly that the 3' non-coding region of the hexon mRNA contains a 609 nucleotide long uninterrupted translational reading frame following a potential initiator AUG. A late 14S mRNA, corresponding to the open reading frame, could be identified by S1 nuclease mapping and electronmicroscopy. The mRNA shares a poly(A) addition site with the hexon and pVI mRNAs, and carries a leader sequence which is related, and probably identical, to the tripartite leader, found in late adenovirus mRNAs. The junction between the leader and the body of this novel mRNA is located within the coding part of the hexon gene.     

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.