Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities.

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular "underlapping," and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24-48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12-14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3 H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus therefore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.     

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.     

Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation.

The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.     

Simian virus 40 host range/helper function mutations cause multiple defects in viral late gene expression.

Simian virus 40 (SV40) deletion mutants dlA2459 and dlA2475 express T antigens that lack the normal carboxy terminus. These mutants are called host range/helper function (hr/hf) mutants because they form plaques at 37 degrees C on BSC-1 and Vero monkey kidney cell lines but not on CV-1p monkey kidney cells. Wild-type SV40 can provide a helper function to permit growth of human adenoviruses in monkey kidney cells; the hr/hf mutants cannot. Progeny yields of hr/hf mutants are also cold sensitive in all cell lines tested. Patterns of viral macromolecular synthesis in three cell lines (Vero, BSC-1, and CV-1) at three temperatures (40, 37, and 32 degrees C) were examined to determine the nature of the growth defect of hr/hf mutants. Mutant viral DNA replication was similar to that of the wild type in all three cell lines, indicating that the mutations affect late events in the viral lytic cycle. In mutant-infected Vero cells, in which viral yields were highest, late mRNA levels were similar to those observed during wild-type infection. Levels of viral late mRNA from mutant-infected CV-1 and BSC-1 cells at 32 and 37 degrees C were reduced relative to those of wild-type-infected cells. The steady-state level of the major viral capsid protein, VP1, in mutant-infected CV-1 cells was reduced to the same extent as was late mRNA. The synthesis of agnoprotein could not be detected in mutant-infected CV-1 cells but was readily detected in CV-1 cells infected by wild-type SV40. Primer extension analyses indicated that most late mRNAs from mutant-infected CV-1 cells utilize start sites downstream from the major wild-type cap site (nucleotide 325) and the agnoprotein initiation codon (nucleotide 335). These results indicate that deletion of the carboxyl-terminal domain of T antigen affects viral late mRNA production, both quantitatively and qualitatively. The agnoprotein is detected late in the wild-type SV40 lytic cycle and is thought to play a role in the assembly or maturation of virions. Reduced hr/hf progeny yields could result from decreased capsid protein synthesis and, in the absence of detectable levels of agnoprotein, from inefficient use of available capsid proteins.     

Polyomavirus small T antigen controls viral chromatin modifications through effects on kinetics of virus growth and cell cycle progression

Minichromosomes of wild-type polyomavirus were previously shown to be highly acetylated on histones H3 and H4 compared either to bulk cell chromatin or to viral chromatin of nontransforming hr-t mutants, which are defective in both the small T and middle T antigens. A series of site-directed virus mutants have been used along with antibodies to sites of histone modifications to further investigate the state of viral chromatin and its dependence on the T antigens. Small T but not middle T was important in hyperacetylation at major sites in H3 and H4. Mutants blocked in middle T signaling pathways but encoding normal small T showed a hyperacetylated pattern similar to that of wild-type virus. The hyperacetylation defect of hr-t mutant NG59 was partially complemented by growth of the mutant in cells expressing wild-type small T. In contrast to the hypoacetylated state of NG59, NG59 minichromosomes were hypermethylated at specific lysines in H3 and also showed a higher level of phosphorylation at H3ser10, a modification associated with the late G(2) and M phases of the cell cycle. Comparisons of virus growth kinetics and cell cycle progression in wild-type- and NG59-infected cells showed a correlation between the phase of the cell cycle at which virus assembly occurred and histone modifications in the progeny virus. Replication and assembly of wild-type virus were completed largely during S phase. Growth of NG59 was delayed by about 12 h with assembly occurring predominantly in G(2). These results suggest that small T affects modifications of viral chromatin by altering the temporal coordination of virus growth and the cell cycle.     

The hr-t gene of polyoma virus

Malignant transformation of cells by polyoma virus results from the continual expression of a viral gene (hr-t) the normal function of which is to facilitate productive viral infection. The series of investigations described here on the polyoma hr-t gene originated with attempts to understand polyoma virus-cell interactions along lines suggested by temperate bacteriophage. Nucleic acid hybridization experiments indicated clearly that viral DNA persists in transformed cells and continues to be expressed. Radiobiological and other experiments, however, suggested a function for the expressed gene(s) which was not expected of a prophage: the promotion, rather than repression, of lytic virus growth. The hr-t gene acts pleiotropically to alter the physiological state of the host in a manner which facilitates virus production and induces a transformed cellular phenotype. The cellular alterations are manifested transiently during productive infection or abortive transformation, but permanently when the viral genome is integrated in stably transformed cells. hr-t mutants are defective in their growth in mice and in most cultured mouse cell lines. They are also unable to induce tumors or any of the morphological, structural, or growth-related changes which accompany cells transformation by the wild-type virus.The 22 kDa and 56 kDa proteins encoded in the early region of the viral DNA constitute dual products of the hr-t gene. hr-t mutants are localized in a narrow segment of the early region that specifies an amino acid sequence shared by these two overlapping proteins. Current efforts to link structural (i.e., mutational) changes with functional changes in these proteins center around the 56 kDa middle T antigen and its associated protein kinase activity. Assayed in vitro, this activity leads to phosphorylation of the 56 kDa protein itself, predominantly at a specific tyrosine residue in the C-terminal portion of the molecule. The middle T protein is anchored in cellular membranes by a hydrophobic tail close to the C-terminus. Membrane association is essential for transformation, as well as for the kinase activity. The common region of the 22 kDa/56 kDa proteins where hr-t mutants map has local regions of homology with highly conserved sequences in the pituitary glycoprotein hormones. The integrity of this region is also essential for transformation and for kinase activity. In vivo, the 56 kDa protein is a substrate for cellular kinase(s) and undergoes multiple phosphorylations (serine and/or threonine) that may affect the tyrosine-specific activity. These kinase reactions, originating in cellular membrane but potentially affecting pathways into the cytoplasm and nucleus, currently provide the most plausible biochemical mechanism underlying the pleiotropic effects of the hr-t gene.     

Tumor antigens induced by nontransforming mutants of polyoma virus.

We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.     

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.     

Temperature-Sensitive Simian Virus 40 Mutant Defective in a Late Function

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.     

A cell regulatory agent, CeReS-18, inhibits mouse 3T6 cell proliferation but not polyomavirus replication

We have purified a cell regulatory sialoglycopeptide, CeReS-18, from intact bovine cerebral cortex cells. This is an 18-kDa molecule that reversibly inhibits cellular DNA synthesis and the proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on mouse 3T6 host cell proliferation and polyomavirus replication was investigated. The results showed that CeReS-18 was able to inhibit 3T6 cell cycling in a concentration-dependent, calcium-sensitive, and reversible manner. Despite the inhibition of cell proliferation, CeReS-18 did not influence polyomavirus infection of 3T6 cells. Indirect immunofluorescent assays revealed that CeReS-18-treated, and cell cycle-arrested, 3T6 cells remained permissive to polyomavirus replication. Electron microscopy and immunogold labeling showed that new viral particles were assembled inside the nuclei of infected cells in the presence of CeReS-18 and during cell cycle arrest. The cellular requirements for the replication of polyomavirus DNA and the synthesis of viral proteins, as well as for the assembly of viral particles, therefore, remained available in CeReS-18-inhibited 3T6 cells. In addition, although polyomavirus infection can be mitogenic, infection of CeReS-18-treated 3T6 cells did not reverse the cell cycle arrest mediated by this cell cycle inhibitor.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Myristylated polyomavirus VP2: role in the life cycle of the virus.

The double-stranded genome of the small DNA tumor virus, polyomavirus, is enclosed in a capsid composed of a major protein, VP1, which associates as pentameric capsomeres into an icosahedral structure, and two minor proteins, VP2 and VP3, whose functions and positions within the structure are unknown. The N-terminal glycine of the VP2 coat protein has been shown to be cotranslationally acylated with myristic acid. To study the function of this modification and the role of VP2 in the life cycle of polyomavirus, the N-terminal glycine, critical to the myristylation consensus sequence, has been altered to a glutamic acid or a valine residue by site-directed oligonucleotide mutagenesis. The glycine----glutamic acid mutant DNA has been further studied. When transfected into cells permissive for the polyomavirus full lytic life cycle, this mutant DNA replicated at levels comparable to those of wild-type viral DNA, and small amounts of nonrevertant (mutant) virus could be harvested from the cultures. The virus particles viewed by electron microscopy appeared slightly distorted, but the ratio of full to empty particles was similar to that produced in a wild-type viral infection. Mutant virus was capable of reinfecting permissive cells but with a considerably reduced efficiency.     

Phosphorylation of polyoma T antigens.

The T antigens of polyoma virus have been examined for phosphorylation in vivo and associated protein kinase activities in vitro. The 100K "large" T antigen is the major phosphoprotein among the T antigen species in vivo as determined by labeling virus-infected cells with 32P-orthophosphate. Hr-t mutants show normal phosphorylation of their 100K T antigens. The wild-type 56K plasma membrane-associated "middle" T antigen is also phosphorylated in the cell, but to a lesser extent than the 100K; this low level phosphorylation is also observed in the presumably altered 56K protein induced by hr-t mutant NG59 and in the 50K truncated "middle" T of hr-t mutant SD15. Addition of dibutyryl cyclic AMP to the medium does not affect labeling of either large or middle T antigens in wild-type- or mutant-infected cells. Thus no differences are observed in T antigen phosphorylation in vivo between wild-type virus and hr-t mutants. Hr-t mutants are defective in a protein kinase activity assayed in vitro by adding gamma-32P-ATP to T antigen immunoprecipitates. In the case of wild-type virus, the 56K protein is the major phosphate acceptor in the in vitro kinase reaction, with a somewhat lower level of phosphorylation observed in the 100K band. Hr-t mutants NG59 and SD15 show no labeling of the altered 56K or 50K, respectively, but do show detectable levels of 32P in the 100K bands. A wild-type virus carrying a small deletion affecting the 100K and 56k bands shows a normal level of kinase activity associated with the truncated T antigens. Ts-a mutants appear to be normal with respect to the middle T antigen-associated kinase. Photoaffinity labeling of infected cell extracts with 8-azido cyclic AMP shows that the two major classes of regulatory subunits of cyclic AMP-dependent protein kinases are present in the immunoprecipitates. Phosphorylation of histone H1 occurs when this substrate is added to immunoprecipitates of either mock-infected or virus-infected cells, again demonstrating the presence of cellular kinases. Further experiments will be required to determine whether the middle T antigen of polyoma virus is itself a protein kinase or simply a substrate for one or more cellular kinases.