Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Effect of a tsA mutation of simian virus 40 late gene expression: variations between host cell lines

Infection of AGMK or CV-1 cells by the early simian virus 40 mutant tsA58 at the permissive temperature (32 degrees C) followed by a shift to the nonpermissive temperature (41 degrees C) caused a substantial decrease in the levels of late viral RNA in the cytoplasm of AGMK cells but not CV-1 cells. At the translational level, this depression of late viral RNA levels was reflected by a decrease in late viral protein synthesis. Thus, in AGMK cells, an early region gene product (presumably large T-antigen) appeared to be continuously required for efficient expression of the late viral genes. In contrast, late simian virus 40 gene expression, once it is initiated in CV-1 cells, continued efficiently regardless of the tsA mutation. The difference in expression of the late simian virus 40 genes in these tsA mutant-infected monkey kidney cell lines may reflect a difference in host cell proteins which regulate viral gene expression in conjunction with early viral proteins.     

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.     

Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40.

Host DNA synthesis is induced when CV-1 (monkey kidney) cell cultures are infected at 40 C with wild-type virions or with temperature-sensitive Simian virus 40 mutants of the "early" complementation group A. Host DNA synthesis is not induced when cultures are infected with mutants of the late complementation group D. The simplest explanation for these observations, that induction depends not upon the expression of some early gene function but rather on the presence of an active D protein in the infecting virion, has been examined. Indirect experiments suggest that this explanation is not correct. Moreover, the induction of host DNA synthesis is impaired when cultures are infected with mutants of the A group at 42.5 C rather than 40 C, suggesting that the A function may be responsible for host induction. The inability of D virions to induce host DNA synthesis may reflect their inability to "uncoat" at 40C.     

Effects of simian virus 40 large and small tumor antigens on mammalian target of rapamycin signaling: small tumor antigen mediates hypophosphorylation of eIF4E-binding protein 1 late in infection

We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.     

Biochemical Consequences of Type 2 Adenovirus and Simian Virus 40 Double Infections of African Green Monkey Kidney Cells

African green monkey kidney (AGMK) cells were nonpermissive hosts for type 2 adenovirus although the restriction was not complete; when only 3 plaque-forming units/cell was employed as the inoculum, the viral yield was about 0.1% of the maximum virus produced when simian virus 40 (SV40) enhanced adenovirus multiplication. The viral yield of cells infected only with type 2 adenovirus increased as the multiplicity of infection was increased. Type 2 adenovirus could infect almost all AGMK cells in culture; adenovirus-specific early proteins and DNA were synthesized in most cells, but small amounts of late proteins were made in relatively few cells. Even when cells were infected with both SV40 and adenovirus, only about 50% were permissive for synthesis of adenovirus capsid proteins. Approximately the same quantity of adenovirus deoxyribonucleic acid (DNA) was synthesized in the restricted as in the SV40-enhanced infection. However, in cells infected with SV40 and type 2 adenovirus, replication of SV40 DNA was blocked, multiplication of SV40 was accordingly inhibited, and synthesis of host DNA was not stimulated. To enhance propagation of type 2 adenovirus, synthesis of an early SV40 protein was essential; 50 mug of cycloheximide per ml prevented the SV40-induced enhancement of adenovirus multiplication, whereas 5 x 10(-6)m 5-fluoro-2-deoxyuridine did not abrogate the enhancing phenomenon.     

Functional genomic mapping of an early-activated centromeric mammalian origin of DNA replication.

Ors12, a mammalian autonomously replicating sequence (812 bp), was previously isolated by extrusion of African green monkey (CV-1 cells) nascent DNA from active replication bubbles. It contains a region of alpha-satellite extending 168-bp from the 5'-end, and a nonrepetitive portion extending from nucleotide position 169 to nucleotide 812 that is present in less than nine copies per haploid genome. Ors12 is capable of transient autonomous DNA replication in vivo and in vitro, associates with the nuclear matrix in a cell cycle-dependent manner, and hybridizes at the centromeric region of six CV-1 cell chromosomes as well as a marker chromosome. To demonstrate that DNA replication initiates at ors12 at a native chromosomal locus, a 14.2 kb African green monkey genomic clone was isolated and sequence information was obtained that allowed us to generate eight sets of PCR primers spanning a region of 8 kb containing ors12. One set of primers occurred inside ors12. These primers were used to amplify nascent DNA strands from asynchronously growing CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR-based mapping methodologies. Both assays showed that DNA replication in vivo initiates preferentially in a 2.3 kb region containing ors12, as well as at a second site located 1.7 kb upstream of ors12. This study provides the first demonstration of genomic function for a centromeric mammalian origin of DNA replication, originally isolated by nascent strand extrusion.     

Identification of postentry restrictions to Mason-Pfizer monkey virus infection in New World monkey cells

TRIM5α has been shown to be a major postentry determinant of the host range for gammaretroviruses and lentiviruses and, more recently, spumaviruses. However, the restrictive potential of TRIM5α against other retroviruses has been largely unexplored. We sought to determine whether or not Mason-Pfizer monkey virus (M-PMV), a prototype betaretrovirus isolated from rhesus macaques, was sensitive to restriction by TRIM5α. Cell lines from both Old World and New World primate species were screened for their susceptibility to infection by vesicular stomatitis virus G protein pseudotyped M-PMV. All of the cell lines tested that were established from Old World primates were found to be susceptible to M-PMV infection. However, fibroblasts established from three New World monkey species specifically resisted infection by this virus. Exogenously expressing TRIM5α from either tamarin or squirrel monkeys in permissive cell lines resulted in a block to M-PMV infection. Restriction in the resistant cell line of spider monkey origin was determined to occur at a postentry stage. However, spider monkey TRIM5α expression in permissive cells failed to restrict M-PMV infection, and interference with endogenous TRIM5α in the spider monkey fibroblasts failed to relieve the block to infectivity. Our results demonstrate that TRIM5α specificity extends to betaretroviruses and suggest that New World monkeys have evolved additional mechanisms to restrict the infection of at least one primate betaretrovirus.     

Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.     

Root hairs and phosphorus acquisition of wheat and barley cultivars

Several genes that restrict nodulation with specific Bradyrhizobiumstrains are known in Glycine max (soybean), and a similar system of nodulation restriction has recently been discovered in the related North American legume Amphicarpaea bracteata. We analyzed how nodulation-restrictive genotypes of each plant interacted with Bradyrhizobium strains sampled from the other host species. Ten bacterial isolates from A. bracteata that nodulated differentially with genotypes of their homologous host legume showed uniform responses to two soybean isogenic lines that differed at the Rj4 locus controlling nodulation restriction: all isolates formed nodules of normal size and morphology on both isolines. However, little or no nitrogen fixation occurred in any of these symbioses. A. bracteata genotypes that displayed broad vs. restricted symbiotic phenotypes toward naturally-associated bradyrhizobia were also tested with two bacterial isolates from soybean (USDA 76 and USDA 123). Both isolates formed nodules and fixed nitrogen in association with both A. bracteata genotypes. However, symbiotic effectiveness (as measured by acetylene reduction assays) was normal only for the combination of USDA 76 with the restrictive A. bracteata genotype. Overall, these results indicate that plant genes that restrict nodulation by certain naturally-associated bradyrhizobia do not confer comparable specificity when plants interact with bacteria from another related legume species.     

Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function

Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.     

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

Surface structure variants inDeinococcus radiodurans

The cell wall morphology and the polypeptide composition of two different strains as well as of two spontaneous mutants ofDeinococcus radiodurans have been compared. The two strains differ with respect to the density of their carbohydrate coat. One of the mutants lacks the surface (HPI) layer; the other one is devoid of a carbohydrate coat.     

Capsid processing requirements for abrogation of Fv1 and Ref1 restriction

Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.     

Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus.

The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base-pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC-1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493-infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread.