Infected Monkey Cells Research Articles

In situ nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site.

New in situ probing methods have been developed and used to probe the nucleoprotein structures at the SV40 major late promoter in infected monkey cells. The region that contains the three proximal transcription elements was probed with DNase I and micrococcal nuclease in transcriptionally active, permeabilized cells, and with the single-strand selective reagent KMnO4 in intact cells. The downstream element is included in a region of enhanced DNase I reactivity at 10- to 11-bp intervals for approximately 140 bp, presumably because of DNA wrapping around a specifically positioned nucleosome particle. The two other proximal DNA elements appear to be mostly melted, with a protecting factor bound primarily to the template DNA strand. The protecting factor directly borders the wrapped particle. These observations provide an initial description of parts of the biological transcription machinery and suggest that the SV40 major late promoter elements are part of a higher order nucleoprotein complex that involves wrapped and melted DNA.

Characterization of the translational defect to fiber synthesis in monkey cells abortively infected with human adenovirus: role of ancillary leaders.

Synthesis of the fiber protein of human adenovirus serotype 2 (Ad2) is 100-fold lower in abortively infected monkey cells, compared with productive infections, despite only a 5- to 10-fold reduction in fiber mRNA levels. Previously Anderson and Klessig (Proc. Natl. Acad Sci. USA 81:4023-4027, 1984) demonstrated a direct correlation between the productive nature of the infection, efficient synthesis of the fiber protein in vivo, and the presence of the x or y ancillary leaders on 10 to 25% of fiber messages. To determine at what level in translation these leaders might be important, the relative rate of initiation and elongation of each class of fiber message was assessed. The presence of the y ancillary leader in productively infected cells increased the rate of initiation about twofold, although translational elongation was similar on all fiber messages. However, the rate of elongation of all fiber messages was threefold slower in abortively infected than in productively infected cells. This reduced elongation rate in abortive infections was specific for fiber. The similar distribution of fiber mRNAs on polysomes in both infections suggests that initiation must also be partially blocked in abortive infections. Since the majority of the fiber mRNA even in productive infections did not contain the ancillary leaders, the initiation and elongation defects in the abortive infection cannot be fully explained by the absence of these leaders. Therefore, other factors in the infected cell must be influencing the rate of translation.

The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen

We have compared the ATPase, DNA-binding, and helicase activities of free simian virus 40 (SV40) large T antigen (To) and T antigen complexed with cellular p53 (T+p53). Each activity is essential for productive viral infection. The T+p53 and To fractions were prepared by sequential immunosorption of infected monkey cells with monoclonal antibodies specific for p53 and T antigen. The immune-complexed T fractions were then assayed in parallel. For ATP hydrolysis, the Vmax for T+p53 was 143 nmol of ADP per min per mg of protein, or 18-fold greater than for To. ATP had no effect on the stability of the T+p53 complex. The T+p53 complex was significantly more active than To in hydrolyzing dATP, dGTP, GTP, and UTP. Of the nucleotide substrates tested, the greatest relative increase (T+p53/To) was in hydrolyzing dGTP and GTP. In DNase footprinting assays performed under replication conditions, the T+p53 complex protected regions I, II, and III of origin DNA while equivalent amounts of To protected only regions I and II. Region III is known to contribute to the efficiency of DNA replication and contains the SP1-binding sites of the early viral promoter. The T+p53 fraction was also a more efficient helicase than To, especially with a GC-rich primer and template. Thus, the T+p53 complex has enhanced ATPase, GTPase, DNA-binding, and helicase activities. These findings imply that complex formation between cellular monkey p53 and SV40 T antigen modulates a number of essential activities of T in SV40 productive infection.

Efficient expression of small RNA polymerase III genes from a novel Simian virus 40 vector and their effect on viral gene expression

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.

Abnormal expression of a late gene family L1 protein in monkey cells abortively infected with adenovirus type 2.

The drastically reduced virus yields obtained from monkey cells abortively infected with adenovirus 2 (Ad2) have been attributed primarily to a severe decrease in the accumulation of the virion protein fiber (IV), a product of the most pomoter distal late gene family, L5. Here we report that the accumulation of virion protein IIIa, a product of the proximal late gene family, L1, is also severely depressed. In contrast, the i-leader protein LO-13.6K and L1 protein(s) 52K/55K are expressed with the same time course and in equal amounts in monkey cells abortively infected by Ad2 or productively infected by the Ad2-simian virus 40 (SV40) hybrid Ad2+ND1 or by the host range mutant Ad2+ND3 hr602. L1-52K/55K is phosphorylated in abortively infected CV-1 or CV-C monkey cells as well as in productively infected human and monkey cells. As with fiber expression, the failure to produce IIIa appears to be due partly to reduced or delayed IIIa mRNA accumulation. The small amount of IIIa protein that is synthesized in monkey cells is stable. Since the accumulation of both IIIa and fiber protein is deficient, the mechanism of abortive infection cannot be attributed solely to the absence of the auxiliary fiber leader sequences (1).

Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen.

pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys----Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplasm of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen.

Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis

At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins all, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32.

Oligomerization and origin DNA-binding activity of simian virus 40 large T antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) exists in multiple molecular forms, some of which are separable by zone velocity sedimentation of soluble extracts from infected monkey cells. Three subclasses of this antigen from SV40-infected monkey cells have been separated and characterized: the 5S, 7S, and 14S forms. Newly synthesized T antigen occurs primarily in the 5S form. Chemical cross-linking provided evidence that the 14S form is primarily a tetramer, whereas the 5S and 7S forms could not be cross-linked into oligomers. The DNA-binding properties of each subclass were investigated after immunopurification. The affinities of the three forms for SV40 DNA and for a synthetic 19-base-pair sequence from binding site I are very similar (equilibrium dissociation constant [KD], 0.3 to 0.4 nM). The specific activity of DNA binding was greatest for the 5S and 7S subclasses and least for the 14S subclass. Moreover, the specific activity of the 5S and 7S subclasses increased sharply at about 40 h after infection, whereas the activity of the 14S subclass was maintained at a constant low level throughout infection. A model relating oligomerization and DNA binding of T antigen in infected cells is presented.

Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40.

Human cells infected with adenovirus type 2 (Ad2) or Ad5 require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state levels of hexon-specific mRNA were found to be comparable in WT- and mutant-infected monkey cells. The in vitro translation experiments showed that double-mutant- and WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs.

Synthesis of the Ad2+ND5-specified 42K protein is regulated posttranscriptionally in abortively infected monkey cells.

In abortive infections of monkey cells by Ad2+ND5, the synthesis of the simian virus 40-specific 42,000-molecular-weight (42K) protein was reduced approximately 10-fold compared with a productive coinfection by Ad2+ND5 plus Ad2hr400 and about 20-fold compared with productive infections by Ad2+ND5 plus simian virus 40 or by Ad2+ND2 alone. However, the level of Ad2+ND5-specific mRNA was depressed twofold or less in abortive infections compared with productive infections. Moreover, the 42K mRNA isolated from abortive Ad2+ND5 infections translated in vitro with the same efficiency as the mRNA isolated from productive coinfections. This is analogous to the block to synthesis of the adenovirus fiber polypeptide in monkey cells (Anderson and Klessig, J. Mol. Appl. Genet. 2:31-43, 1983). Also like fiber protein, the increased level of the 42K protein found in productive infections was due to enhanced synthesis, not increased stability of the protein. Our results suggest that the synthesis of the Ad2+ND5-specified 42K protein and the adenovirus fiber protein are regulated in similar posttranscriptional manners.

Microinjection of mRNA enhances translational efficiency of human adenovirus fiber message in monkey cells.

In monkey cells abortively infected with human adenovirus serotype 2, the synthesis of the fiber polypeptide of the virion capsid is reduced by at least a factor of 100 when compared with that in monkey cells productively infected with a host range mutant of adenovirus serotype 2 (Ad2hr400). However, the steady-state level of fiber-encoding mRNA present in abortively infected monkey cells is only reduced by a factor of 5 to 10. When mRNA isolated from abortively and productively infected monkey cells was microinjected into the cytoplasms of uninfected or abortively infected monkey cells, no differences in the efficiency of translation of the fiber messages from these two sources were observed. These results suggest that the block to synthesis of the fiber polypeptide in abortively infected monkey cells does not reside in the translational machinery of the abortively infected cells themselves but may involve compartmentalization of the fiber message within the cells or an altered processing of the fiber message which prevents correct presentation to the ribosomes.

Microinjection of mRNA enhances translational efficiency of human adenovirus fiber message in monkey cells.

In monkey cells abortively infected with human adenovirus serotype 2, the synthesis of the fiber polypeptide of the virion capsid is reduced by at least a factor of 100 when compared with that in monkey cells productively infected with a host range mutant of adenovirus serotype 2 (Ad2hr400). However, the steady-state level of fiber-encoding mRNA present in abortively infected monkey cells is only reduced by a factor of 5 to 10. When mRNA isolated from abortively and productively infected monkey cells was microinjected into the cytoplasms of uninfected or abortively infected monkey cells, no differences in the efficiency of translation of the fiber messages from these two sources were observed. These results suggest that the block to synthesis of the fiber polypeptide in abortively infected monkey cells does not reside in the translational machinery of the abortively infected cells themselves but may involve compartmentalization of the fiber message within the cells or an altered processing of the fiber message which prevents correct presentation to the ribosomes.

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin

The antitumor drug, m-AMSA (4′-(9-acridinylamino)-methanesulfon-m-anisidide), is known to interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the enzyme-DNA complex in its putative cleavable state. Treatment of SV40 virus infected monkey cells with m-AMSA resulted in both single- and double-stranded breaks on SV40 viral chromatin. These strand breaks are unusual because they are covalently associated with protein. Immuno-precipitation results suggest that the covalently linked protein is DNA topoisomerase II. These results are consistent with the proposal that the drug action in vivo involves the stabilization of a cleavable complex between topoisomerase II and DNA in chromatin. Mapping of these double-stranded breaks on SV40 viral DNA revealed multiple topoisomerase II cleavage sites. A major topoisomerase II cleavage site was preferentially induced during late infection and was mapped in the DNAase I hypersensitive region of SV40 chromatin.

Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs

To examine the role of small nuclear ribonucleoproteins (snRNPs) in mRNA splicing, we have injected SV40 DNA, in the presence or absence of anti-Sm or anti-(U1)RNP antibodies, into the nucleus of X. laevis oocytes, and analyzed the viral specific RNAs and proteins that were synthesized. In the absence of antibodies, the majority of the viral mRNAs were spliced, giving rise to transcripts and proteins analogous to those found in infected monkey cells. However, the relative efficiencies with which the various splice sites were utilized were different in the two cell types. When sera from systemic lupus erythematosus (SLE) patients containing anti-Sm or anti-(U1)RNP antibodies were coinjected with the viral DNA, splicing of L-strand-specific (late) mRNA was dramatically inhibited. Cleavage at both 5′ and 3′ splice sites was blocked, leading to an accumulation of unspliced primary transcripts. Neither the total amount of late RNA synthesized nor the formation of mature polyadenylated late mRNA 3′ ends was affected. These results indicate that U1 snRNPs play a crucial role in mRNA splicing in vivo. Unexpectedly, the effects of the sera on E-strand-specific (early) viral mRNA splicing were different. All anti-Sm or -(U1)RNP sera tested had no detectable effect on the splicing of the mRNA coding for the small tumor antigen. A subset of these sera, however, inhibited large tumor antigen mRNA splicing. On the basis of these data it is suggested that different pre-mRNAs, or even different splice sites within the same pre-mRNA, have dissimilar interactions with snRNP particles in the splicing reaction.

Stepwise phosphorylation of the NH2-terminal region of the simian virus 40 large T antigen.

The simian virus 40 (SV40) large T antigen was immunoprecipitated from extracts of infected monkey cells and cleaved with trypsin under conditions of mild proteolysis. The digestion generated fragments from the NH2-terminal region of T antigen which were released from the immunoprecipitates. Pulse-chase experiments showed that most of the newly made T antigen (form A) generated an NH2-terminal fragment of 17 kDa in size, whereas most of the T antigen that had aged in the cell (form C) generated a fragment of 20 kDa. An intermediate form of T antigen (form B), which generated an 18.5- kDa NH2-terminal fragment, was produced in part from form A and was converted to form C during the chase. Phosphate-labeling experiments showed that form C was the species of T antigen that incorporated the most 32P radioactivity at the NH2-terminal region, although some label was also incorporated into forms A and B. In vitro dephosphorylation of gel-purified 18.5- and 20-kDa fragments labeled with [35S]methionine increased the electrophoretic mobility of the fragments to that of 17 kDa. This signified that phosphorylation of the NH2-terminal fragments was directly responsible for their aberrant behavior in acrylamide gels. Although peptide maps of the methionine-labeled tryptic peptides of the 17-, 18.5-, and 20-kDa fragments were very similar to one another, maps of the 32P-labeled tryptic Pronase E peptides of these fragments contained qualitative and quantitative differences. Analysis of the labeled phosphoamino acids of various peptides from these fragments indicated that the 20-kDa fragment was highly phosphorylated at Ser 123 and Thr 124, whereas the 17- and 18.5-kDa fragments were mostly unphosphorylated at these sites. These experiments indicated that T antigen is phosphorylated at the NH2-terminal region in a specific stepwise process and, therefore, that this post-translational modification of T antigen is tightly regulated.

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Messenger RNA encoding the fiber protein of the human adenovirus serotype 2 (Ad2) capsid is inefficiently translated in abortively infected African green monkey kidney cells. The amount of fiber mRNA present in the cytoplasm of abortively infected monkey cells is less than that in productively infected cells by a factor of 5-10 but synthesis of the fiber polypeptide is reduced by a factor of more than 100. Evidence from a variety of experiments indicates that the defect does not lie in the translational apparatus of the monkey cell but may best be explained by differences in the fiber messages made in abortively versus productively infected cells. Here we report that fiber mRNA isolated from abortively infected monkey cells is processed differently than that made in productively infected cells. Primer extension analysis of the 5' ends of fiber messages from several different productive and abortive infections shows a direct correlation between synthesis of the fiber polypeptide in vivo and the presence of the "x" and/or "y" ancillary leaders on messages encoding the fiber polypeptide. Of all the mRNAs encoded by the major late transcriptional unit of Ad2 only the fiber message can contain the x and y leaders, and the fiber protein is the only late Ad2 protein reported to be glycosylated. We speculate that these leader sequences play a role in the synthesis of this glycoprotein, as well as that of the Ad2 19-kilodalton glycoprotein encoded by early region 3, whose mRNA also contains the x and y leaders.

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.