Isolation of vaccinia virus mutants capable of replicating independently of the host cell nucleus

alpha-Amanitin-resistant vaccinia virus mutants were isolated after serial viral passages in BSC-40 cells that were carried out in the presence of inhibitory levels (6 micrograms/ml) of alpha-amanitin. One such mutant, alpha-27, was highly refractory (greater than 95%) to alpha-amanitin-mediated inhibition and was selected for further study. In the absence of drug, the phenotypes of alpha-27 and wild-type vaccinia virus were indistinguishable with respect to growth kinetics. DNA synthesis, protein synthesis, and morphogenesis. Infections in the presence of alpha-amanitin revealed two striking differences, however. First, wild-type virus was unable to catalyze proteolytic processing of the two major capsid proteins VP62 and VP60, whereas alpha-27 was most efficient at this process. Second, wild-type viral morphogenesis within the infected cells was arrested by alpha-amanitin at an apparently analogous step to that previously described for enucleated cells. This observation was supported by the ability of alpha-27 virus to replicate in enucleated BSC-40 cells. Restriction enzyme analyses of alpha-27 versus wild-type genomes revealed that a XhoI cleavage site was altered in the alpha-27 DNA molecule, suggesting a possible location for the alpha-amanitin resistance locus.     

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.     

Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.     

Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication.

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.     

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.     

Heterogeneity of the 5'' terminus of late mRNA induced by a viable simian virus 40 deletion mutant.

d1-1811 is a viable simian virus 40 deletion mutant which lacks the DNA region corresponding to the major capping site of the late viral RNA. The exact size of the deletion (40 base pairs) was determined by comparison of the mutant DNA sequence with the wild-type simian virus 40 (strain 776) DNA sequence. Although d1-1811 forms somewhat smaller plaques, the amount of viral RNA late after infection was not significantly reduced compared with that of the wild type. Virus-specific, polyadenylate-containing, 32P-labeled late RNA was purified from the cytoplasm and enzymatically degraded to characterize the 5' terminus. The cap-containing oligonucleotides were isolated, and their structures were analyzed by further digestion. Instead of a single cap structure, we found a variety of capped 5' termini, with adenosine caps occurring much more frequently than guanosine caps. Nevertheless, there was a remarkable homology between both types of terminal sequences. Conceivably, the minor cap population present in wild-type simian virus 40 late mRNA may correspond to the collection of capped termini identified in the d1-1811 late mRNA . Cellular cytoplasmic RNA shows a similar pattern of cap structures, but the relative abundance is quite different.     

Identification of a tolerant locus on Arabidopsis thaliana to hypervirulent beet curly top virus CFH strain

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted boting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.     

p53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G₁. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.     

A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly

Mutations introduced into the capsid gene of duck hepatitis B virus (DHBV) were tested for their effects on viral DNA synthesis and assembly of enveloped viruses. Four classes of mutant phenotypes were observed among a series of deletions of covering the 3' end of the capsid open reading frame. Class I mutant capsids were able to support normal single-stranded and relaxed circular viral DNA synthesis; class II mutant capsids supported normal single-stranded DNA synthesis but not relaxed circular DNA synthesis; class III mutant capsids resembled class II capsids, but viral DNA synthesis was inhibited 5- to 10-fold; and class IV capsids were severely restricted in their ability to support viral DNA synthesis. Class I capsids were assembled into enveloped virions, but class II, III, and IV capsids were not. Viral DNA synthesized inside class II capsids was normal with respect to minus-strand DNA initiation, plus-strand DNA initiation, and circularization of the DNA, but plus strands failed to be elongated to mature 3-kb DNA. The results suggest that a function of the capsid protein specifically required for viral DNA maturation is also required for assembly of nucleocapsids into envelopes. Thus, class II mutants appear to be defective in the appearance of the "packaging signal" for virus assembly (J. Summers and W. Mason, Cell 29:403-415, 1982).     

Synthesis of hepadnavirus particles that contain replication-defective duck hepatitis B virus genomes in cultured HuH7 cells.

To evaluate the possibility of producing transducible replication-defective hepadnaviruses, cloned mutant duck hepatitis B virus genomes were tested both for virus antigen production and viral DNA synthesis following transfection into the human hepatoma cell line HuH7. Deletion of a cis-acting 12-nucleotide sequence implicated in viral DNA synthesis, direct repeat 1 (DR1), resulted in the loss of ability to synthesize both mature viral DNA and infectious virus. The delta DR1 mutant, however, produced envelope and core antigens and was shown to provide trans-acting functions required for the assembly of infection-competent particles. Thus, mutants with mutations in viral genes could be rescued as DNA-containing viral particles after cotransfection with delta DR1. The efficiency of rescue was influenced by the site of mutation. A mutant DNA encoding truncated core and envelope proteins not only was poorly rescued but also was able to suppress the production from a wild-type DNA of infectious virus.     

Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants.

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.     

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.

A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.     

Murine teratocarcinoma: A model for virus-cell interaction in a differentiating cell system

The stem cell of the murine teratocarcinoma is refractory to infection with Simian virus 40 and polyoma. Utilizing various procedures, we attempted to alter this block to infection by modifying the infection procedure. Multiple infections with high-titer SV₄₀ and pretreatment of cells with DEAE-dextran or the carcinogen 4-nitroquinoline l-oxide did not induce embryonal carcinoma cells to produce T- antigen. Co-infection with adenovirus 5, which infects the embryonal carcinoma, and SV₄₀ did not induce the expression of SV₄₀ Tantigen. Therefore, these procedures did not overcome the block to virus infection. The assay for the SV₄₀ T antigen was immunofluorescence; however, the immunoprecipitation technique did not detect T antigen in the infected embryonal carcinoma cells. Finally, the viral DNA present in the embryonal carcinoma was examined for its ability to replicate. These studies showed that viral DNA was not replicating as assayed by the viral DNA's sensitivity to UV irradiation when replicating in the presence of 5-bromodeoxyundine.     

Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10(5) to 10(6) of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.     

Abortive infection with Sindbis virus of a Chinese hamster ovary cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis

The effects of phosphatidylserine starvation on the infection with Sindbis virus (an enveloped RNA virus) have been investigated in a Chinese hamster ovary (CHO) cell mutant (strain PSA-3) which requires exogenously added phosphatidylserine for cell growth because it lacks the ability to synthesize this phospholipid. When PSA-3 cells were grown in the absence of phosphatidylserine, the cellular contents of phosphatidylserine and also phosphatidylethanolamine produced through decarboxylation of phosphatidylserine decreased. Sindbis virus production in the mutant cells decreased immediately upon phosphatidylserine deprivation as did the contents of phosphatidylserine and phosphatidylethanolamine, whereas the cell growth, viability, and syntheses of protein, DNA and RNA remained normal for approx. 40 h phosphatidylserine starvation. Although PSA-3 cells grown without phosphatidylserine for 24 h were able to bind and internalize Sindbis virus almost normally, viral RNA synthesis was greatly reduced in the cells, suggesting that nucleocapsids of internalized Sindbis virus are not normally released into the cytoplasm. Unlike mammalian cell mutants defective in endosomal acidification, PSA-3 cells grown without phosphatidylserine were not resistant to diphtheria toxin. Furthermore, the yield of virions and viral RNA synthesis in PSA-3 cells were not completely restored on brief exposure of the cells to low pH medium following virus adsorption, which is known to induce artificial fusion of the viral envelope with the plasma membrane of normal host cells and then injection of viral nucleocapsids into the cytoplasm. Our data demonstrate the requirement of membrane phospholipids, such as phosphatidylserine and/or phosphatidylethanolamine, in CHO cells for Sindbis virus infection, and we discuss their possible roles.     

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.     

Cis effect of the type 5 adenovirus E1A gene enhancer element on cellular transformation

Mutants of type 5 adenovirus that lack all or part of the early region 1A (E1A) gene enhancer element transform rodent embryo fibroblast (CREF) cells at higher efficiencies than wild-type virus. An analysis of viral E1A cytoplasmic mRNA levels in mutant and wild-type virus-infected CREF cells revealed no differences in the levels of the E1A mRNAs. This implies that a decrease in the rate of viral E1A gene expression was not responsible for the transforming properties of the enhancer-less viruses. Unlike wild-type virus, however, the mutant viruses were able to replicate their genomes in the normally nonpermissive CREF cells. This change in viral DNA template concentration further resulted in an increase in early gene mRNA concentrations in mutant-virus-infected CREF cells. These studies suggest several possible mechanisms that could be responsible for the increased transforming potentials of these viruses, including 1) a cis effect of removing the viral E1A enhancer element on the efficiency of viral DNA integration, 2) viral DNA replication, or 3) an increase in the levels of the viral E1A and E1B mRNAs owing to viral DNA replication in the virus-infected CREF cells.