DNA binding properties and replication activity of the T antigen related D2 phosphoprotein

According to earlier genetic experiments, a region within the N-terminal 50-100 amino acids may be important for the replication function of T antigen, the initiator protein of simian virus 40 (SV40). We have investigated this possibility using the T antigen related D2 protein in several biochemical assay systems. D2 protein, a phosphoprotein coded for by the adeno-SV40 hybrid virus Ad2+D2, shares its 594 C-terminal amino acids with authentic T antigen and its 104 N-terminal amino acids with an adenovirus structural protein. We confirmed earlier studies showing that D2 protein appeared to bind well to specific binding sites in the SV40 origin of replication. We found, however, that D2 protein was rather inefficient, inducing the unwinding of the double-stranded origin region, and was much less active than authentic T antigen as an initiator of in vitro SV40 DNA replication. We interpret these findings to indicate that D2 protein molecules associate with the origin to form an aberrant complex that is quite inefficient, inducing DNA unwinding and the establishment of replication forks. The possibility that the N-terminus may be required for an optimal arrangement of T antigen at the origin was supported by results of dephosphorylation studies. Dephosphorylation of N-terminal phosphoamino acids had significant effects on the stability of D2 protein-origin complexes.     

Immunological Cross-Reactivity Between Simian Virus 40 Large T Antigen and D2 Hybrid T Antigen

A specific antiserum was raised in rabbits against D2 hybrid T antigen that had been purified from HeLa cells infected with the adenovirus/simian virus 40 hybrid, Ad2(+)D2. The specificity of this serum was compared with that of a conventional hamster antiserum against simian virus 40-induced tumors by immunoprecipitation and by a new radioimmune assay that can detect nanogram quantities of D2 hybrid T antigen.     

Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.     

Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen

A quantitative, enzyme-linked immunoadsorbent assay has been developed for the simian virus 40 large T antigen. When hamster anti-simian virus 40 tumor serum was used, this method permitted specific identification of large T antigen and its analog, the D2 hybrid protein, a molecule with the same C-terminal approximately 600 amino acids as large T antigen. The sensitivity limit of this test was 0.63 ng of protein. The slopes of the regression lines of the enzyme-linked immunosorbent assay titrations performed with highly purified D2 or simian virus 40 large T antigen and with crude extracts of simian virus 40-infected monkey and transformed human cells were identical. Thus, the curve generated with a purified protein, such as D2, can serve as a quantitative standard for the measurement of large T antigen in a wide variety of extracts. Furthermore, solutions containing high salt concentrations and buffers containing up to 0.1% Nonidet P-40 did not interfere with the assay, making it applicable to the measurement of large T antigen in a variety of chromatographic fractions. The enzyme-linked immunosorbent assay was three times more sensitive, was significantly faster to perform, and was quantitatively valid over a much broader large-T-antigen concentration range than the complement fixation test. As such, it should be useful in future studies of the structure and function of this protein.     

Cell surface T antigen in cells infected with simian virus 40 or an adenovirus-simian virus 40 hybrid, Ad2+D2.

Cell surface T antigen, detected by a radioimmune assay that uses 125I-labeled Staphylococcus aureus protein A and antibodies against either authentic T antigen or D2 hybrid T antigen, was found in simian virus 40-transformed and -infected cells and in cells infected with an adenovirus-simian virus 40 hybrid, Ad2+D2. In simian virus 40 lytic infection, the surface T antigen appeared at the same time as the nuclear T antigen.     

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.     

Context affects nuclear protein localization in Saccharomyces cerevisiae.

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.     

Monoclonal antibodies against simian virus 40 tumor antigens: analysis of antigenic binding sites, using adenovirus type 2-simian virus 40 hybrid viruses.

The antigenic binding sites of two monoclonal antibodies are located in the COOH-terminal region (clone 412) and probably in an internal region (clone 7) of simian virus 40 large T antigen. A third monoclonal antibody (clone 122), which has been shown to bind nonviral T antigen, does not react with HeLa cells infected with nondefective adenovirus type 2 (Ad2)-simian virus 40 hybrid viruses Ad2+ND1, Ad2+ND2, or Ad2+ND4.     

Genomic arrangement of an adenovirus-simian virus 40 hybrid virus, Ad2+ND4del.

Ad2+ND4del is an adenovirus type 2-simian virus 40 hybrid virus nondefective for growth in human cells. The virus was first observed when stocks of Ad2+ND4, a hybrid isolated from primary monkey kidney cells, were propagated in human cells. This paper describes the DNA sequence at two sites of DNA recombination, the site of the left adenovirus type 2-simian virus 40 junction and the site of a deletion of internal simian virus 40 sequences. Since the deletion was observed when the virus was switched from monkey to human cells, an analysis of gene expression in the region of DNA rearrangement may prove useful for the elucidation of molecular events that accompany virus growth in different hosts.     

Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G.

We constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middle-size tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.     

DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene

We describe a 1635-bp Herpes simplex virus type 2 (HSV-2) DNA sequence containing the entire coding region of glycoprotein D (gD-2). The amino acid sequence of gD-2, deduced from the nucleotide sequence, was compared to that of the analogous Herpes simplex virus type 1 (HSV-1) glycoprotein (gD-1). The two glycoproteins are 85% homologous and contain highly conserved regions of as much as 49 amino acids in length. Comparison of DNA sequences upstream from gD-1 and gD-2 coding regions identified possible conserved regulatory sequences.     

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.     

Biosynthesis, Immunological Specificity, and Intracellular Distribution of the Simian Virus 40-Specific Protein Induced by the Nondefective Hybrid Ad2+ND1

Ad2(+)ND(1), a nondefective hybrid virus containing a segment of the early region of simian virus 40 (SV40) DNA covalently inserted into the human adenovirus 2 genome, enhances the growth of human adenoviruses in simian cells and induces the SV40 U antigen. This hybrid previously has been shown to code for a 28,000 (28K) molecular weight protein not present in wild-type adenovirus 2-infected cells. By radioimmunoprecipitation using sera from hamsters bearing SV40-specific tumors, we have established that the Ad2(+)ND(1)-induced 28K protein is SV40-specific. This Ad2(+)ND(1)-induced protein is synthesized as a 30K molecular weight precursor, which is detectable only when infected cells are pulse-labeled in the presence of the protease inhibitor tosylamino phenylethyl chloromethyl ketone. Upon fractionation of labeled cell extracts, about 80% of the 28K protein is found in the plasma membrane fraction, whereas the remaining 20% is associated with the outer nuclear membrane. This protein is not detectable either in the nucleus or in the cytoplasm. Blockage of proteolytic cleavage by tosylamino phenylethyl chloromethyl ketone did not alter the topographic distribution of this SV40-specific protein, although the amount of the precursor protein in the outer nuclear membrane increased fourfold while that in the plasma membrane was proportionately decreased. This result suggests that the 28K protein is transferred from the outer nuclear membrane to the plasma membrane after posttranslational cleavage of the 30K precursor polypeptide. These data offer further support to the proposal that the 28K protein contains the determinants for SV40 U antigen and is responsible for SV40 enhancement of adenovirus growth in simian cells.     

N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.     

Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.     

New properties of simian virus 40 large T antigen

An 8,000-molecular-weight (8K) T antigen was found in all cells transformed by simian virus 40. The 8K T antigen was weakly labeled in vivo with [35S]methionine or 32Pi. A deletion in the human papovavirus BK genome, in the region coding for the carboxy-terminal end of the large T antigen, reduced the size of the 8K T antigen. The last 80 amino acids of the large T antigen include the sequence Asp-Asp-Asp-Asp unique to the activation peptide of trypsinogen. Large T antigen bound diisopropyl fluorophosphate and was retained by D-phenylalanine coupled to Sepharose beads, an affinity adsorbent that can retain chymotrypsin. The large T antigen and the recA protein of Escherichia coli, a known protease, have several properties in common as well as several similar sequences. Antibodies against large T antigen interacted with native recA protein.     

Simian virus 40 tumor antigen: isolation of the origin-specific DNA-binding domain

To localize the origin-specific DNA-binding domain on the simian virus 40 tumor (T) antigen molecule, we used limited proteolysis with trypsin to generate fractional peptides for analysis. A 17,000-Mr peptide was found to be capable of binding not only to calf thymus DNA, but also specifically to the simian virus 40 origin of DNA replication. This approximately 130-amino-acid peptide was derived from the extreme N-terminus of the T antigen and represented less than one-fifth of the entire molecule. The coding sequence for this tryptic peptide was located approximately between 0.51 and 0.67 map units (excluding the intron, which maps between 0.54 and 0.59). Since the first 82 amino acids are shared between large T and small t antigens, and since the latter does not bind DNA, it can be concluded that the sequence between isoleucine 83 and approximately arginine 130 is necessary for origin-specific binding by the T antigen. We also observed that in vivo phosphorylation of the T antigen within this region completely abolished the ability of the 17,000-Mr peptide to bind DNA. This observation is consistent with the idea that DNA binding by the T antigen is regulated by posttranslational modifications.     

Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.     

Mapping of the viral mRNA encoding a super-T antigen of 115,000 daltons expressed in simian virus 40-transformed rat cell lines

Simian virus 40-transformed V11 F1 clone 1 subclone 7 rat cells produced a considerable amount of an elongated form of large-T antigen with an Mr of 115,000 (115K super-T antigen), but these cells did not produce detectable traces of normal-sized large-T antigen (86,000 daltons) (P. May, M. Kress, M. Lange, and E. May, Cold Spring Harbor Symp. Quant. Biol. 44:189-200, 1980). First, a comparison of the tryptic peptide fingerprints of 115K super-T and large-T antigens suggested that 115K super-T antigen is simian virus 40 coded and contains a duplication of amino acid sequences of large-T antigen. Second, from S1 mapping analysis of 115K super-T mRNA, performed with various restriction fragments of simian virus 40 DNA, it was concluded that super-T mRNA is a form of large-T mRNA containing a tandem duplication of the sequence extending from approximately 0.46 to 0.35 map unit. The duplicated sequence corresponded to that region of the simian virus 40 genome in which 12 of 13 tsA mutation sites are clustered (C. J. Lai and D. Nathans, Virology 66:70-81, 1975).