Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.     

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.     

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.     

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.     

A novel 39-kilodalton membrane protein binds GTP in polyomavirus-transformed cells.

To investigate the possible involvement of GTP-binding proteins in transformation by the DNA tumor virus polyomavirus, the GTP-binding activities of Ras-like proteins and G protein alpha subunit proteins were examined in polyomavirus-transformed cells. No differences in the degrees or patterns of expression of Ras-like proteins were observed. However, a 39-kDa protein specifically bound GTP in membranes from polyomavirus-transformed cells. This protein was not seen in nontransformed or lytically infected cells or in phenotypically normal revertants of polyomavirus-transformed cells. It reappeared, however, in spontaneous retransformants derived from the revertants. The 39-kDa protein was not found stably associated with polyomavirus T antigens, nor was it phosphorylated on tyrosine. The 39-kDa protein was not recognized by an antiserum specific for members of the Gi alpha subfamily of G proteins or by antisera against all other known GTP-binding proteins of similar molecular mass. These results suggest that this novel 39-kDa GTP-binding membrane protein is observed as part of a long-term response that accompanies stable transformation by the virus.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

Octyl-beta-D-glucopyranoside extracts polyomavirus receptor moieties from the surfaces of mouse kidney cells.

Polyomavirus receptor moieties were extracted from the surfaces of mouse kidney cells with the nonionic detergent octyl-beta-D-glucopyranoside. Following extraction with this detergent, mouse kidney cells were refractory to polyomavirus infection. Binding studies demonstrated that this loss of susceptibility resulted from extraction of a peripheral membrane protein or proteins required for proper virus attachment to and infection of mouse kidney cells. Infection of extracted mouse kidney cells returned following a 2-h recovery period. However, the presence of cycloheximide or tunicamycin in the recovery media interfered with recovery from infection. Cells could be infected immediately after extraction by supplying them with the extracted moieties prior to or concomitant with infection. A complex of polyomavirus and the extracted receptor protein was formed by in vitro incubation and was stable in sucrose gradient analysis. Functional receptor moieties were prepared in the form of liposomes from the detergent extract. The virus-receptor complex was immunoprecipitated with anti-polyomavirus immunoglobulin G, and the portion of the complex contributed by the cell was identified. Immunoblot analysis of the mouse kidney cell detergent extract with a receptor-specific 125I-labeled anti-idiotypic antibody or 125I-labeled polyomavirus demonstrated several reactive proteins. Attachment of polyomavirus to mouse kidney cells, followed by extraction of the virus-receptor complex, identified polyomavirus-binding proteins similar to those observed in in vitro binding. Proteins with molecular weights of approximately 95,000, 50,000 and 25,000 to 30,000 were consistently observed in all receptor assays. The relationship between these proteins and their possible involvement as the cell receptor for polyomavirus are discussed.     

Transformation by hamster polyomavirus: identification and functional analysis of the early genes.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.     

Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse fibroblasts.

Transfection of 3T6 cells with a cloned polyomavirus genome encoding only large T antigen resulted in DNA replication with only about 1/10 the efficiency of wild-type viral DNA coding for all three T antigens. This replication defect was at least in part overcome by the simultaneous transfections of polyomavirus genomes which allowed the expression of small T antigen. We conclude that polyomavirus small T antigen has a (probably indirect) role in replication.     

Cytologic and biomolecular diagnosis of polyomavirus infection in urine specimens of HIV-positive patients

OBJECTIVE: To evaluate the frequency of human polyomavirus reactivation in urine specimens from HIV-positive patients; compare the sensitivity of cytology, immunohistochemistry and molecular biology; differentiate viral genotypes; and correlate the results with urinary cytologic abnormalities. STUDY DESIGN: Urine specimens from 78 unselected HIV-positive patients were evaluated by means of cytology, immunohistochemistry and nested polymerase chain reaction (n-PCR) to evaluate the presence of polyomaviruses. Restriction fragment length polymorphism (RFLP) was carried out in positive cases in order to differentiate BK virus (BKV) from JC virus (JCV). CD4 cells and serum creatinine levels were evaluated as indices of immune status and renal function, respectively, whereas the presence of red blood cells was used as an index of urogenital damage. RESULTS: Cytologic evidence of polyomavirus infection was found in 17 samples and immunohistochemically confirmed in 9; another 6 cytologically negative cases were detected by means of immunohistochemistry. In all cases, only one or two cells showed typical viral inclusions or positive staining. n-PCR identified 44 positive samples, thus confirming all of the cytologically and immunohistochemically positive cases and detecting polyomavirus genome in a further 21. RFLP detected 39 JCV, 1 BKV and 4 JCV-BKV infections. No correlation was found between the presence or type of polyomavirus and immune status, but red blood cells were found more frequently in the positive than in the negative samples. Serum creatinine levels fell within the normal range in all cases. CONCLUSION: Molecular biology is the most sensitive tool for detecting polyomavirus urinary infection in HIV-positive patients and the only reliable method of differentiating JCV and BKV viral genotypes.     

Early steps of polyomavirus entry into cells

The mechanism by which murine polyomavirus penetrates cells and arrives at the nucleus, the site of viral replication, is not well understood. Simian virus 40 and JC virus, two closely related members of the polyomavirus subfamily, use caveola- and clathrin-mediated uptake pathways for entry, respectively. The data presented here indicate that compounds that block endocytosis of both caveola- and clathrin-derived vesicles have no effect on polyomavirus infectivity. Polyomavirus does not appear to colocalize with either clathrin light chain or caveolin-1 by immunofluorescence microscopy. Additionally, expression of a dominant-negative form of dynamin I has no effect on polyomavirus uptake and infectivity. Therefore, polyomavirus uptake occurs through a class of uncoated vesicles in a clathrin-, caveolin-1-, and dynamin I-independent manner.     

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.     

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly.

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.     

Cellular ras gene activity is required for full neoplastic transformation by polyomavirus.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.