The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.     

Simian virus 40 production after viral uncoating in the CV-1 cell nucleus.

A normal yield of infectious simian virus 40 was produced by a CV-1 cell culture after inoculation of a few cells with an average of one particle per nucleus by microinjection.     

Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants

Summary SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37° C, but give at least 10 000-fold lower yields than wild type virus in BSC cells at 32° C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein throught to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation. This work was supported by grants CA40586 and BRSG 2S07RR07084-23 to J. M. P. and grant CA33079 to L. T., from the National Institutes of Health, Bethesda, MD.     

Suppression of a VP1 mutant of simian virus 40 by missense mutations in serine codons of the viral agnogene.

We isolated second-site revertants of a partially defective VP1 mutant of simian virus 40. The suppressing mutation in each of these pseudorevertants was mapped to the viral agnogene. Of six independently isolated pseudorevertants, all had a missense mutation in a serine codon, near the beginning of the agnogene, that would cause replacement of serine at position 7, 11, or 17 in the agnoprotein by a hydrophobic amino acid. Our results suggest that the agnoprotein interacts in a specific way with VP1 during the late stages of viral development.     

Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin.

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.     

Isolation and characterization of simian virus 40 early region deletion mutants.

We constructed a tsB4/dl884 double-mutant helper virus and used it to isolate two simian virus 40 early region deletion mutants that lack about half of the DNA sequences normally used to encode the large tumor antigen (T). Both mutants make a normal-sized small t antigen, but neither mutant can replicate its DNA in the absence of a T+ helper.     

Hormonal regulation of the transformation phenotype in simian virus 40-transformed rat embryonic preadipose cell lines.

The regulation of transformed phenotypes was studied in newly isolated preadipose cell lines which were established after infection with simian virus 40 tsA58 dl2009. The clonal cell lines isolated exhibited most of the characteristics typical of transformed cells. The transformants, however, were able to differentiate into adipocytes in the presence of low calf serum (0.5%) and a combination of several hormones, including hydrocortisone and insulin. Treatment with insulin alone stimulated the growth of these cells but did not induce lipid accumulation without added hydrocortisone. The effect of hydrocortisone was accompanied by a restoration of growth control in the transformants after they reached high cell density. The blot hybridization analysis of cellular DNAs digested by restriction enzymes revealed that simian virus 40 genomes were integrated at multiple separate sites at which a head-to-tail oligomeric insertion took place. Large T antigen was synthesized in growing cells but was regulated at high cell density when cells were committed to differentiate by glucocorticoids. These results suggest that the glucocorticoid hydrocortisone is capable of restoring growth regulation at high cell densities to simian virus 40-transformed preadipose cell lines.     

Construction and analysis of viable deletion mutants of simian virus 40.

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.     

Missense mutations in the VP1 gene of simian virus 40 that compensate for defects caused by deletions in the viral agnogene.

Simian virus 40 mutants lacking sequences in the late leader region are viable but produce smaller plaques than does wild-type virus. Within three passages at low multiplicities of infection, virus stocks of several such mutants accumulated variants that synthesized an altered form of the major virion protein, VP1, having a slightly faster mobility in sodium dodecyl sulfate-polyacrylamide gels than did the wild-type protein. Because these variants overgrew the original virus stocks, we consider them to be second-site revertants. By construction and characterization of a series of recombinants, the second-site mutations were shown to map to at least two different regions of the VP1 gene. Nucleotide sequence analysis indicated that single-amino-acid changes were responsible for the rapid mobility of VP1. When combined in cis with either a wild-type or mutant leader region, these VP1 mutations sped up by 10 to 20 h the time course of accumulation of infectious progeny but not of viral DNA or VP1. LP1, the protein encoded by the agnogene, was shown previously to be necessary for the efficient transport of the virion proteins to the nucleus or for their efficient assembly with viral minichromosomes. The VP1 missense mutations reported here compensate for the lack of LP1 by facilitating this process. On the basis of these findings and findings reported previously by us and others, we hypothesize that LP1 facilitates the formation of infectious particles by inhibiting the polymerization of VP1 molecules until the time they interact with viral minichromosomes; the VP1 mutations reported here compensate for the loss of LP1 by lessening the potential of VP1 for self-polymerization.     

Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells.

A new method was developed for isolation of intracellular forms of simian virus 40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the infectious cycle. In contrast to the Triton extraction method, which yields only a 60-70S complex, this new procedure yielded three forms of SV40 nucleoprotein complexes: complex I, complex II, and the nature virion (V). The three nucleoprotein complexes differed in physical as well as biochemical properties. Complex I, which is only a small portion of the total SV42 nucleoprotein complexes late during infection, was active in synthesizing both SV40-specific DNA and RNA. Pulse-labeling experiments suggest the following metabolic pathway: I leads to II leads to V. Conversion of complex I to II occurred shortly after the completion of SV40 DNA replication and resulted in the inactivation of the biosynthetic activities of I.     

Characterization of simian virus 40 transformed African green monkey cells (CV-1). I. Defective virion and viral genome.

Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens.     

Role of the agnoprotein in regulation of simian virus 40 replication and maturation pathways.

Analysis of two agnogene mutants, dl2304 deleted over the entire agnogene and in2379 carrying a 2-base insert, indicated that the mutant phenotype of small plaque formation must be the result of a defect late in the maturation pathway. Both mutants were removed from the pool of molecules available for replication with wild-type kinetics. Whereas dl2304 was somewhat reduced in its rate of progression from chromatin to previrions-virions, in2379, which produced even smaller plaques than dl2304 did, progressed with wild-type kinetics. Therefore, the agnoprotein was not required for progression from chromatin to previrions.     

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.     

Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.

We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.     

Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.     

Transformation-defective mutants of feline sarcoma virus which express a product of the viral src gene.

Mink cell cultures infected with the Snyder-Theilen strain of feline sarcoma-leukemia virus were cloned from single cells under conditions favoring single virus-single cell interactions. The primary colonies included (i) typical feline sarcoma virus (FeSV)-transformed nonproducer clones, one of which segregated revertants, and (ii) FeSV-infected, phenotypically normal clones, three of which spontaneously converted to the transformed phenotype. The revertants and spontaneous transformants were compared with parental and sister clones expressing the opposite phenotype. Transformed subclones formed colonies in agar, were tumorigenic in nude mice, and failed to bind epidermal growth factor, whereas flat sister subclones were indistinguishable from uninfected mink cells in each of these assays. Sister subclones derived from the same infectious event contained FeSV proviruses integrated at the same molecular site, regardless of which phenotype was expressed. One revertant clone, however, lacked most FeSV proviral DNA sequences but retained terminal portions of the FeSV genome which persisted at the original site of proviral DNA insertion. Two flat subclones expressed viral RNA and the phosphorylated "gag-x" polyprotein (pp78gag-x) encoded by the gag and src sequences of the FeSV genome. Both of these clones were susceptible to retransformation by FeSV. Although unable to induce foci, the viruses rescued from these cells contained as much FeSV RNA as the focus-forming viruses rescued from transformed sister subclones and could be retransmitted to mink cells, again inducing FeSV gene products without signs of morphological transformation. We conclude that these FeSV genomes represent transformation-defective mutants.     

Stimulation of the acute-phase response in simian virus 40-hepatocyte cell lines.

Seven simian virus 40 (SV40)-hepatocyte cell lines were characterized with respect to the ability to express eight liver acute-phase genes. cDNA clones corresponding to albumin, serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, alpha-, beta-, and gamma-fibrinogen, and alpha 1-major-acute-phase protein mRNAs were used in Northern (RNA) or slot blot analyses. In the noninduced state, six of the seven cell lines showed significant (i.e., liverlike) levels of constitutive expression of all genes examined except that expression of haptoglobin mRNA was considerable lower than in the normal liver. To examine whether these immortalized liver cells can respond appropriately to inflammatory mediators, cells were treated with conditioned medium from activated human monocytes or mixed lymphocyte cultures. Results showed that these SV40-hepatocyte cell lines responded to the conditioned media in culture by down-regulating albumin gene expression and up-regulating other acute-phase genes in a time- and dose-dependent manner. These results indicate that the SV40-hepatocytes retained not only the ability to express a number of acute-phase genes but also the ability to respond to external stimuli. The usefulness of these cell lines for analysis of the molecular mechanisms involved in the regulation of these acute-phase genes is discussed.     

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.