Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.     

Analysis of simian virus 40 wild-type and mutant virions by agarose gel electrophoresis.

Intact wild-type simian virus 40 particles can be separated and resolved from a temperature-sensitive mutant and from a number of other viruses by agarose gel electrophoresis. The relative mobilities of the viruses appear to be a function of both virion size and surface composition. The virions of a temperature-sensitive strain of simian virus 40, tsB204, have significantly greater mobility than those of wild-type simian virus 40, when electrophoresis was conducted toward the cathode at pH 5.0. When electrophoresis was performed toward the anode at pH 7.0, TSB204 viruses have a slightly slower mobility as compared with that of the wild type. The data indicated that the virions of tsB204 have a greater positive charge at their surface than those of wild-type particles. No differences were detected in the finger print patterns of the tryptic peptides of VP1 and VP3 of these two virus strains. Although it was not possible to identify the structural polypeptide directly affected by the tsB204 mutation, we have shown that this mutation affects charge distribution on the surface of the virion.     

Recurring defective variants of simian virus 40 containing monkey DNA segments.

Four independently and newly isolated defective variants of simian virus 40 have been characterized. All four are very similar, if not identical, to two previously and independently isolated variants (Wakamiya et al., J. Biol. Chem. 254:3584-3591, 1979; J. Papamatheakis, E. Kuff, E. Winocour, and M. F. Singer, J. Biol. Chem. 255:8919-8927, 1980). The documented similarities include restriction endonuclease maps and the presence of the same monkey DNA segments covalently linked to simian virus 40 DNA sequences. Each of the newly described variants was first detected upon serial passaging of wild-type simian virus 40 at a high multiplicity of infection at 33 degrees C as recently described (M. F. Singer and R. E. Thayer, J. Virol. 35:141-149, 1980). A variety of experiments support the idea that the various isolates were independent and do not reflect inadvertent cross-contamination. Two of the new isolates arose during passage of wild-type strain 777 virus in BSC-1 cells, one during passage of strain 776 in BSC-1 cells, and one during passage of strain 776 in primary African green monkey kidney cells. The two variants obtained after passage of strain 776 were shown to contain a particular recognition site for restriction endonuclease MboII within their simian virus 40 DNA segments, as do the two previous isolates. This site is not present in wild-type strain 776 DNA but is shown here to be present in wild-type strain 777 DNA. The surprising recurrence of closely related variants and particularly the unexpected presence of the endo R.MboII site in variants derived from passaging strain 776 suggest that these variants may arise by mechanisms other than recombination between the initial infecting viral genome and the host DNA.     

A simian virus 40 dl884/tsA58 double mutant is temperature sensitive for abortive transformation

We have constructed a dl884/tsA58 double mutant and a t+T- early-region deletion mutant and have used these mutants to study the roles of the simian virus 40 tumor antigens (T and t) in transformation. Our major conclusions are that (i) although the mutant tsA58 is not temperature sensitive for the maintenance of transformation, the dl884/tsA58 double mutant is; (ii) small t antigen can provide at least one, but not all, of the functions required for the maintenance of transformation; and (iii) at least two different functions are required for the maintenance of simian virus 40 transformation.     

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.     

Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation.

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.     

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.     

The nucleotide sequence of repetitive monkey DNA found in defective simian virus 40.

DNA fragments containing monkey DNA sequences have been isolated from defective SV40 genomes that carry host sequences in place of portions of the SV40 genome. The fragments were isolated by restriction endonuclease cleavage and contain segments homologous to sequences in both the highly repetitive and unique (or less repetitive) classes of monkey DNA. The complete nucleotide sequence of one such fragment [151 base pairs (bp)] predominantly homologous to the highly reiterated class of monkey DNA was determined using both RNA and DNA sequencing methods. The nucleotide sequence of this homogeneous DNA segment does not contain discernible multiple internal repeating units but only a few short oligonucleotide repeats. The reiteration frequency of the sequence in the monkey genome is >10⁶. Digestion of total monkey DNA (from uninfected cells) with endonuclease R Hind III produces relatively large amounts of discrete DNA fragments that contain extensive regions homologous to the fragment isolated from the defective SV40 DNA.A second fragment, also containing monkey sequences, was isolated from the same defective substituted SV40 genome. The nucleotide sequence of the 33 bp of this second fragment that are contiguous to the 151 bp fragment has also been determined.The sequences in both fragments are also present in other, independently derived, defective substituted SV40 genomes.     

Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments.

Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent.     

A cis-acting DNA signal for encapsidation of simian virus 40.

Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the sesdeleted plasmid 2 kbp away from the ori, suggesting that for encapsidation the two DNA elements have to be close to each other. These findings afford novel strategies for the investigation of viral encapsidation.     

trans-dominant defective mutants of simian virus 40 T antigen.

We constructed a collection of linker insertion mutants in the simian virus 40 (SV40) genome and studied several of these with changes limited to a part of the large T antigen gene corresponding to an amino acid sequence shared with other ATPases. Two of these mutants were found to have a novel phenotype in that they could not be complemented for plaque formation by a late-region deletion mutant. These two mutants, in contrast to other mutants in this region, were able to transform rat cells in culture at a frequency close to that of the wild-type gene. The noncomplementing mutants were found to be potent inhibitors of SV40 DNA replication despite the presence of wild-type T antigen in the transfected cells. This inhibition was shown to be the result of the introduced mutations in the large T antigen gene. We conclude that the large T antigens of the noncomplementing mutants can act as inhibitors of SV40 DNA replication.     

Endonucleases and simian virus 40 virions: origin of a virion-associated endonuclease.

The origin and role of the endonuclease activity associated with purified virions of simian virus 40, previously described by this and other laboratories, have been further investigated. We found that the enzymatic activity from virions of temperature-sensitive (ts) mutants is not more heat labile than that from wild-type virions. This result was obtained for a variety of ts mutants, including three of the tsA class, and in experiments in which the enzyme was tested in both the presence and absence of viral particles. Comparison of the viron enzyme with endonucleases prepared from either serum or nuclei of uninfected cells reveals a similarity between the viron and serum enzymes based on chromatographic behavior and relative activity with different cations. Virus particles prepared free of this endonuclease were still infectious. We were unsuccessful in uncovering endonuclease in such preparations upon disruption. These data emphasize the necessity for caution in interpreting the role of particle-associated enzymes.     

Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production.     

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts.

We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.     

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.