DNA-binding region of the simian virus 40 tumor antigen.

The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular-weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment-binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA.     

Cold-sensitive regulatory mutants of simian virus 40

A preparation of short synthetic myosin filaments (minifilaments) in the absence of other myosin forms is reported. Myosin minifilaments have been prepared by dialysing myosin from vertebrate striated muscle into 10 mm-citrate/Tris buffer (pH 8.0 at 4 °C) containing no other salt. These polymers of myosin are very stable and show little tendency to aggregate or dissociate in the original solvent. Sedimentation velocity, diffusion and viscosity measurements indicate that the minifilaments are composed of 16 to 18 molecules. Examination of electron micrographs reveals that the bare central region of minifilaments extends over 1600 to 1800 Å and the entire particles are about 3000 Å long with a diameter of 80 Å across the smooth region. They have the appearance of short bipolar filaments (Huxley, 1963). In solution the minifilaments are homogeneous in terms of size distribution and exhibit normal MgATPase and CaATPase activities. When examined in the ultracentrifuge, the minifilaments sediment in the form of a hypersharp peak (or bar) with a sedimentation coefficient independent of rotor speed. The minifilaments can be dissociated by ATP, hardly by MgATP; whereas KCl (between 0.04 and 0.2 m) induces further polymerization. It is suggested that the minifilaments are an intermediate in the assembly of myosin filaments.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

Rearrangement of simian virus 40 regulatory region is not required for induction of progressive multifocal leukoencephalopathy in immunosuppressed rhesus monkeys

Rearrangements of the JC virus (JCV) regulatory region (RR) are consistently found in the brains of patients with progressive multifocal leukoencephalopathy (PML), whereas the archetype RR is present in their kidneys. In addition, the C terminus of the large T antigen (T-Ag) shows greater variability in PML than does the rest of the coding region. To determine whether similar changes in simian virus 40 (SV40) are necessary for disease induction in monkeys, we sequenced the SV40 RR and the C terminus of the T-Ag from the brain of simian/human immunodeficiency virus (SHIV)-infected monkey 18429, which presented spontaneously with an SV40-associated PML-like disease, as well as from the peripheral blood mononuclear cells (PBMC), kidneys, and brains of SV40-seronegative, SHIV-infected monkeys 21289 and 21306, which were inoculated with the 18429 brain SV40 isolate. These animals developed both SV40-associated PML and meningoencephalitis. Thirteen types of SV40 RR were characterized. Compared to the SV40 archetype, we identified RRs with variable deletions in either the origin of replication, the 21-bp repeat elements, or the late promoter, as well as deletions or duplications of the 72-bp enhancer. The archetype was the most prominent RR in the brain of monkey 18429. Shortly after inoculation, a wide range of RRs could be found in the PBMC of monkeys 21289 and 21306. However, the archetype RR became the predominant type in their blood, kidneys, and brains at the time of sacrifice. On the contrary, the T-Ag C termini remained identical in all compartments of the three animals. These results indicate that unlike JCV in humans, rearrangements of SV40 RR are not required for brain disease induction in immunosuppressed monkeys.     

Naturally arising recombinants that are missing portions of the simian virus 40 regulatory region.

When simian virus 40 (SV40) is serially passaged at high multiplicity, a heterogeneous collection of naturally arising variants is generated. Those which are the most abundant presumably have a selective replicative advantage over other defective and wild-type helper SV40s. Two such naturally arising host-substituted variants of SV40 have been characterized in terms of complete nucleotide sequence determination. Evolutionary variant ev-1101 (previously isolated by Lee et al., Virology 66:53-69, 1975) is from undiluted serial passage 13, whereas ev-2101 is newly isolated from undiluted serial passage 6 of an independently-derived evolutionary series. Both variants contain a five-times tandemly repeated segment of DNA consisting of viral Hin C and Hin A sequences that have recombined with a segment of host DNA that is not highly reiterated in the monkey genome. The monkey segment differs in the two variants as does the size of the viral segment retained. In two additional host-substituted variants, ev-1102 (previously isolated from serial passage 20 by Brockman et al., Virology 54:384-397, 1973) and ev-1108 (newly isolated from serial passage 40), the SV40 sequences derived from the replication origin are present as inverted repetitions. The inverted repeat regions of these two variants have been analyzed at the nucleotide sequence level and are compared with SV40 variant ev-1104 from passage 45 (previously characterized by Gutai and Nathans, J. Mol. Biol. 126:259-274, 1978). The viral segment containing the regulatory signals for replication and viral gene expression is considerably shortened in later serial passages as demonstrated by these five variants. It is of interest that the variants presumably arose due to their enhanced replication efficiency, yet are missing some of the sequence elements implicated in the regulation of replication. Furthermore, a comparison of the structure of the replication origin regions indicates that additional changes occur in the SV40 regulatory region with continued undiluted serial passage.     

Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Adoptive transfer assays performed in this laboratory have shown that peritoneal exudate cells from 10-day primiparous hamsters are cytotoxic to SV40-transformed sarcoma cells (WF5-1) carrying fetal antigen, whereas peritoneal exudate cells from multiparous hamsters are less cytotoxic. This suggests a suppressor activity might be present during subsequent pregnancies that reduces the responsiveness of lymphocytes from pregnant hamsters to stimulation by fetal antigens on tumor cells. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi- and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.     

The simian virus 40 large tumor antigen

In this review, I hope to achieve the following: (a) to document the presence of a lysosome-like proton pump ATPase in many different membrane systems of animal, plant and microbial origin; (b) to glean from the diverse data common characteristics of these ATPases, especially as regards their similarities and differences with mitochondrial-type F1F0 proton pump ATPases; and (c) to consider questions of synthesis and regulation of a cellular proton pump system with such a widespread distribution.     

Inactivation of adenovirus and simian virus 40 tumorigenicity in hamsters by vaccine processing methods

The effectiveness of the adenovirus vaccine inactivation process in destroying the tumorigenic potential for hamsters of adenoviruses, simian virus 40 (SV-40), and adenovirus-SV-40 hybrids was studied. Baby hamsters injected with untreated virus and with samples subjected to the complete inactivation process and to portions of the process were observed for tumor development for periods in excess of 300 days. Over 20,000 hamsters were injected. From 1 to 7 hr of exposure to formaldehyde at a concentration of 0.031 m at 37 C was sufficient to destroy the tumorigenicity observed in the nontreated preparations. Since the inactivation process included 48 hr of exposure at 37 C to 0.031 m formaldehyde plus treatment with ultraviolet (UV) and with beta-propiolactone (BPL), it was concluded that the process has a large margin of safety. Adenovirus isolates free from tumorigenic potential are difficult, if not impossible, to obtain. Therefore, a proven inactivation process appears to provide the best assurance for obtaining adenovirus vaccines free from such potential. Data presented suggest that the tumorigenic property of the viruses studied might be independent of the infectivity of the preparation. The tumorigenic property was found to be highly susceptible to formaldehyde, but less sensitive to BPL or UV treatment. In contrast, treatment with UV or BPL decreased viral infectivity more readily than tumorigenicity. The three-stage inactivation process (formaldehyde, UV, and BPL) inactivated both tumorigenicity and infectivity.     

Action spectrum for the in vitro induction of simian virus 40 by ultraviolet radiation

A line of simian virus 40-transformed hamster kidney cells was exposed to ultraviolet radiation at eleven different wavelengths in the region 238-302 nm. An action spectrum derived from the resulting exposure-response curves for the induction of simian virus 40 from these cells exhibits a broad peak in the region 260-270 nm suggesting DNA as the major chromophore for this response. This conclusion is consistent with results obtained by other investigators who have noted viral induction by a number of DNA-damaging agents.     

Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.

In an attempt to distinguish simian virus 40 (SV40) large T antigen (T) binding to ATP from hydrolysis, specific mutations were made in the ATP-binding site of T according to our model for the site (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Two acidic residues predicted to make contact with the magnesium phosphate were changed to alanines. The mutated T gene was completely defective for viral DNA synthesis and for virion production, and it was dominant defective for viral DNA replication. The defective T gene encoded a stable product (2905T) that oncogenically transformed mouse cell lines. 2905T, immunoprecipitated from transformed-cell extracts, bound SV40 origin DNA specifically and, surprisingly, it was active as an ATPase. A recombinant baculovirus was constructed for the production and purification of the mutant protein for detailed biochemical analyses. 2905T had only 10% of the ATPase and helicase of wild-type T. The Km of 2905T for ATP in ATPase assays was the same as the Km of wild-type T. ATP activated the ATPase activity of wild-type T, but not of 2905T. As tested by gel bandshift assay, 2905T bound to SV40 origin DNA and to individual sites I and II with affinities similar to that of the wild type. However, ATP did not modulate the DNA-binding activity of mutant T to site II. Therefore, this mutation in the ATP-binding site in T resulted in defects in the interaction between the protein and ATP that appeared to be responsible for the determination of the active state of T for DNA binding versus ATPase.     

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate stimulates simian virus 40 induction by DNA-damaging agents and tumor initiators.

Simian virus 40 (SV40)-transformed Syrian hamster kidney cells produce infectious SV40 virus particles after treatments which damage DNA, such as UV irradiation or mitomycin C treatment. We have found that the induction of SV40 by DNA-damaging agents is greatly stimulated when a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), is present in the medium. Phorbol, which has a molecular structure similar to TPA but does not have any tumor-promoting activity, showed no such stimulatory effect on SV40 induction. This apparent synergistic effect of DNA-damaging agents and tumor promoter (TPA) was more pronounced when a tumor initiator, benzo [a]pyrene or 2-acetamido-fluorene, was combined with TPA. The effect of TPA on UV-triggered SV40 induction was greatly influenced by the timing of TPA addition to the culture medium, which was most efficient when addition of TPA was 5 to 20 h before UV irradiation. The effect of TPA, however, was not observed in SV40 rescue from hamster cells by cell fusion with permissive monkey (C7) cells.     

Tumor induction by simian virus 40 in mice is controlled by long-term persistence of the viral genome and the immune response of the host.

Simian virus 40 (SV40), which transforms mouse cells in vitro, has not been previously observed to cause tumors when injected in immunocompetent mice. We have investigated both the fate of the injected virion in mice and several immunological parameters as potential factors controlling tumorigenicity. We find that although SV40 does not replicate in mouse cells, the viral DNA can persist for many months postinjection; the majority of the viral DNA is found in the cytoplasm, but a small amount of the viral DNA is integrated at multiple sites in the host nuclear DNA. The persistence of the viral genome is independent of the ability of the mouse to mount an SV40 TSTA specific cytotoxic T-cell response and may be attributed to the cytoplasmic location of the majority of the viral genome. However, in long-term studies of SV40-injected mice, genetically identical except for the major histocompatibility complex, we find that tumors were induced in some mice of the H-2d (low cytotoxic T-lymphocyte responder to SV40 TSTA) but not of the H-2k (high responder to SV40 TSTA) haplotype. Thus, a combination of inefficient disposal of the injected virion and inefficient immunological surveillance and elimination of cells containing nuclear SV40 DNA can eventually result in SV40-induced tumors at multiple sites in mice.     

Intracellular localization of viral polypeptides during simian virus 40 infection.

African green monkey kidney cells infected by simian virus 40 were analyzed by immunofluorescence techniques for the nature and the time course of the appearance of viral polypeptides during infection. Reagents used in the study were anti-Vpl sera and affinity-purified anti-Vpl immunoglobulin G, anti-Vp3 sera, antivirus (anti-V) sera, and anti-tumor antigen sera. The results are summarized as follows. (i) Three types of staining, nuclear, perinuclear, and perinuclear accompanied by cytoplasmic staining, were observed in infected cells in reaction with anti-vpl antibody. In addition, a highly structured staining was observed at the periphery of nuclei of infected cells late in infection. (ii) In reaction with anti-Vp3 serum, the staining was confined within nuclei of cells throughout infection. (iii) Vp1 and Vp3 antigens seem to occupy different spacial regions of the nuclear area in cells. (iv) Vp1 and Vp3 antigens were expressed simultaneously during infection. (v) Centriolar staining observed early in infection paralleled the appearance of tumor (T-) antigen until 24 h after infection, after which time the frequency of positive centriolar staining decreased as infection progressed. (vi) T-antigen was first expressed at about 8 h after infection, and Vp1 and Vp3 antigens were first expressed at about 20 h after infection.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Assemblages of simian virus 40 capsid proteins and viral DNA visualized by electron microscopy

SV40 assembles in the nucleus by addition of capsid proteins to the minichromosome. The VP15VP2/3 capsomer is composed of a pentamer of the major protein VP1 complexed with a monomer of a minor protein, VP2 or VP3. In the capsid, the capsomers are bound together via their flexible carboxy-terminal arms. Our previous studies suggested that the capsomers are recruited to the packaging signal ses via avid interaction with Sp1. During assembly Sp1 is displaced, allowing chromatin compaction. Here we investigated the interactions in vitro of VP1(5)VP2/3 capsomers with the entire SV40 genome, using mutant VP1 deleted in the carboxy-arm that cannot assemble, but retains DNA-binding capacity. EM revealed that VP1(5)VP2/3 complexes bind non-specifically at random locations around the DNA. Sp1 was absent from mature virions. The findings suggest that multiple capsomers attach simultaneously to the viral genome, increasing their local concentration, facilitating rapid, concerted assembly reaction and removal of Sp1.     

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.