Phenotype-specific phosphorylation of simian virus 40 tsA mutant large T antigens in tsA N-type and A-type transformants.

To identify molecular differences between simian virus 40 (SV40) tsA58 mutant large tumor antigen (large T) in cells of tsA58 N-type transformants [FR(tsA58)A cells], which revert to the normal phenotype after the cells are shifted to the nonpermissive growth temperature, and mutant large T in tsA58 A-type transformants [FR(tsA58)57 cells], which maintain their transformed phenotype after the temperature shift, we asked whether the biological activity of these mutant large T antigens at the nonpermissive growth temperature might correlate with phosphorylation at specific sites. At the permissive growth temperature, the phosphorylation patterns of the mutant large T proteins in FR(tsA58)A (N-type) cells and in FR(tsA58)57 (A-type) cells were largely indistinguishable from that of wild-type large T in FR(wt648) cells. After a shift to the nonpermissive growth temperature, no significant changes in the phosphorylation patterns of wild-type large T in FR(wt648) or of mutant large T in FR(tsA58)57 (A-type) cells were observed. In contrast, the phosphorylation pattern of mutant large T in FR(tsA58)A (N-type) cells changed in a characteristic manner, leading to an apparent underphosphorylation at specific sites. Phosphorylation of the cellular protein p53 was analyzed in parallel. Characteristic differences in the phosphorylation pattern of p53 were observed when cells of N-type and A-type transformants were kept at 39 degrees C as opposed to 32 degrees C. However, these differences did not relate to the different phenotypes of FR(tsA58)A (N-type) and FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature. Our results, therefore, suggest that phosphorylation of large T at specific sites correlates with the transforming activity of tsA mutant large T in SV40 N-type and A-type transformants. This conclusion was substantiated by demonstrating that the biological properties as well as the phosphorylation patterns of SV40 tsA28 mutant large T in cells of SV40 tsA28 N-type and A-type transformants were similar to those in FR(tsA58)A (N-type) and in FR(tsA58)57 (A-type) cells, respectively. The phenotype-specific phosphorylation of tsA mutant large T in tsA A-type transformants probably is a cellular process induced during establishment of SV40 tsA A-type transformants, since tsA28 A-type transformant cells could be obtained by a large-T-dependent in vitro progression of cells of the tsA28 N-type transformant tsA28.3 (M. Osborn and K. Weber, J. Virol. 15:636-644, 1975).     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

Properties of permissive monkey cells transformed by UV-irradiated simian virus 40.

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.     

Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.     

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.     

Accumulation of cells with 4N DNA content at nonpermissive temperature in rat embryo diploid cells transformed by tsA mutant of simian virus 40

Primary rat embryo cells were transformed by a tsA mutant (tsA640) of simian virus 40 (SV40). Proliferation of all four independent diploid transformants was suppressed at a nonpermissive temperature (40.3 degrees C), being accompanied by a marked increase in the fraction of cells with a 4N DNA content (a 4N peak in the flow cytofluorogram). However, in this case, the fraction of cells with a 2N DNA content (a 2N peak in the flow cytofluorogram) was preserved. Both effects (suppression of proliferation and increase in the 4N peak) diminished when transformed cells were superinfected with wild-type SV40. The increased 4N peak was preserved, albeit not completely, for at least 24 hours, when cells were further incubated in the presence of hydroxyurea at the nonpermissive temperature. On the other hand, the preserved 2N peak all but disappeared within 24 hours, when cells were further incubated in the presence of colcemid at the nonpermissive temperature. These results suggest that the thermolabile large T antigen of SV40 directly or indirectly induces an accumulation of cells with a 4N DNA content, at the nonpermissive temperature, by prolonging the G2 (and/or late S) period.     

Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mutant revertants.

The growth properties of hamster cells transformed by wild-type Simian virus 40 (SV40), by early SV40 temperature-sensitive mutants of the A complementation group, and by spontaneous revertants of these mutants were studied. All of the tsA mutant-transformed cells were temperature sensitive in their ability to form clones in soft agar and on monolayers of normal cells except for CHLA-30L1, which was not temperature sensitive in the latter property. All cells transformed by stable revertants of well-characterized tsA mutants possessed certain growth properties in common with wild-type-transformed cells at both temperatures. Virus rescued from tsA transformants including CHLA30L1 was temperature sensitive for viral DNA replication, whereas that rescued from revertant and wild-type transformants was not thermolabile in this regard. T antigen present in crude extracts of tsA-transformed cells including CHLA30L1, grown at 33 degreeC, was temperature sensitive by in vitro immunoassay, whereas that from wild-type-transformed cells was relatively stable. T antigen from revertant transformants was more stable than the tsA protein. Partially purified T antigen from revertant-transformed cells was nearly as stable as wild-type antigen in its ability to bind DNA after heating at 44 degrees C, whereas T antigen from tsA30 mutant-transformed cells was relatively thermolabile. These results further indicate that T antigen is a product of the SV40 A gene. Significantly more T antigen was found in extracts of CHLA30L1 grown to high density at the nonpermissive temperature than in any other tsA-transformed cell similarly grown. This is consistent with the suggestion that the amount of T antigen synthesized in CHLA30L1 is large enoughto allow partial expression of the transformed phenotype at the restrictive temperature. Alternatively, the increase in T antigen concentration may be secondary to one or more genetic alterations that independently affect the transformed phenotype of these cells.     

Alteration in cyclic adenosine 3':5'-monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature-sensitive mutant (tsA640) of SV40

By using a photoaffinity ligand, cell extracts from transformed macrophages that were established by infection with temperature-sensitive mutants (tsA640) of simian virus 40 (SV40) were examined for cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins. At the nonpermissive temperature for SV40 large T antigen, 39.0 degrees C, no significant cAMP-binding proteins could be detected, such as primary mouse macrophages. At the permissive temperature of 33.0 degrees C, cAMP-binding proteins appeared later than SV40 T antigen expression and cellular DNA synthesis. The profile of cAMP-binding proteins was similar to that of resting, but not proliferating, mouse clonal fibroblasts (BALB/c 3T3). These and previous results suggest that SV40 T antigen influences the expression of cAMP-binding proteins in tsA640-transformed macrophages; the large/small T antigen converts the profile of cAMP-binding proteins from macrophage to fibroblastic cells.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. I. Rescue of a Simian Virus 40 Temperature-Sensitive Mutant by Passage in Permissive Transformed Monkey Lines

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.     

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.     

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.     

Induction of fibronectin expression, actin cable formation, and entry into S phase following reexpression of T antigen in mouse macrophages transformed by the tsA640 mutant of SV40

Mouse macrophages transformed by a temperature-sensitive mutant (tsA640) of simian virus 40 (SV40) were examined by immunofluorescence microscopy for fibronectin expression and actin distribution. Resting cultures of tsA640 transformants incubated at a temperature nonpermissive for SV40 large T antigen (39.0 degrees C) exhibited phagocytic activity and did not exhibit cellular fibronectin and actin cables, like primary cultures of resident macrophages. When the resting cultures were sparsely seeded and shifted down to the permissive temperature of 33.0 degrees C, expression of large T antigen in the nucleus, expression of fibronectin in the cytoplasm, and cellular entry into S phase occurred in that temporal order, followed by actin cable formation, cellular proliferation, and diminishment of phagocytic activity. The expression of T antigen and fibronectin was sensitive to actinomycin D and cycloheximide. The expression of fibronectin was insensitive to inhibitors of DNA synthesis, whereas the expression of actin cables was sensitive. These results suggest that SV40 T antigen leads macrophages to express fibronectin and actin cables, as well as resumption of cell proliferation, and that entry into S phase is not required for fibronectin expression but may be required for actin cable formation.     

Temperature dependency for maintenance of transformation in mouse cells transformed by simian virus 40 tsA mutants.

Mouse embryo fibroblasts and 3T3 cells were transformed by wild-type, tsB4, tsA7, tsA58, and tsA209 simian virus 40. Clones of transformants were generated both in soft agar and in liquid medium by focus formation and at both high and relatively low multiplicities of infection. All transformants were assayed for three phenotypes of transformation: (i) the ability to form highly multinucleated cells in cytochalasin B-supplemented medium, i.e., uncontrolled nuclear division; (ii) the capacity to continue DNA synthesis at increasing cell density; and (iii) the ability to form colonies in soft agar. The great majority of mouse embryo fibroblast transformants generated with tsA mutant virus were temperature sensitive for transformation in all three assays, regardless of the input multiplicity or whether they were generated in liquid medium or soft agar. These transformants exhibited a normal or near-normal phenotype at the nonpermissive temperature of 40 degrees C. All but one of the transformants which appeared transformed at both temperatures were in the A209 group. In contrast to mouse embryo fibroblasts, transformants generated with 3T3 cells and tsA virus were often not temperature sensitive, exhibiting the transformation phenotypes at both temperatures. This phenomenon was more often observed when 3T3 transformants were generated in soft agar. These results, along with other published data, suggest that uncontrolled nuclear division and uncontrolled DNA synthesis are a function of the simian virus 40 A gene. Finally, with the 3T3 transformants, there was often discordance in the expression of transformation among the three phenotypes. Some tsA transformants were temperature sensitive in one of two assays but were transformed at both 33 and 40 degrees C in the remaining assay(s). Other transformants exhibited a normal cytochalasin B response at either temperature but were temperature sensitive in the other assays.     

Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

Replicative intermediates in UV-irradiated simian virus 40

We have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [3H]thymidine, and isolated by centrifugation in CsCl/ethidium bromide gradients followed by BND-cellulose chromatography. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h, by which time the size of the newly-synthesized DNA exceeded the interdimer distance. No significant excision of dimers from parental strands in either replicative intermediates or Form I (closed circular) DNA molecules was detected. These data are consistent with the hypothesis that replication forks are temporarily blocked by dimers encountered on the leading strand side of the fork, but that daughter strand continuity opposite dimers is eventually established. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strands contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from UV-damaged SV40 replicative intermediates.