Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis.

Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.     

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Temperature-Sensitive Mutants of Adenovirus Type 12 Defective in Viral DNA Synthesis

Ten temperature-sensitive (ts) mutants of adenovirus type 12 which produce plaques at 31 but not at 38.5 C have been isolated after mutagenesis with nitrosoguanidine or nitrous acid. The mutants have been classified into six separate complementation groups. DNA-DNA hybridizations have shown that at 38.5 C the ts 401 and 406 mutants of groups B and E, respectively, synthesized less than 10% of the normal level of viral DNA. The two mutants were also defective in the production of late proteins at the nonpermissive temperature, as shown by fluorescent-antibody tests and analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Genetic recombination between the ts viruses 401 and 406 has been demonstrated; the recombination frequency for the wild-type virus production was 17.7%. Both mutants induced an increase in thymidine kinase activity at 38.5 C. Moreover, the two viral DNA-defective mutants shut off host DNA synthesis at the restrictive temperature. It is striking that at 38.5 C ts virus 401 transformed two to eight times more hamster cells than the wild-type virus, whereas ts virus 406 transformed at a frequency similar to the wild-type virus.     

DNA strand exchange catalyzed by proteins from vaccinia virus-infected cells.

Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex. The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product. Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps. The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa. The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+. One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing. As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors. Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail. The recovery of these structures shows that the enzyme catalyzes true strand exchange. There is also a unique polarity to the strand exchange reaction. The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand. Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product. Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.     

Isolation and characterization of a heat-inducible simian virus 40 mutant.

We have isolated a new type of temperature-sensitive mutant of simian virus 40 (SV40) that is capable of productive infection in permissive cells but not of maintenance of viral DNA integration in transformed cells at the conditional temperature. Virus development is induced when cells transformed by this mutant are shifted to temperatures above 39 degrees C, but is not induced below this temperature. The plaque-purified, temperature-sensitive mutant virus confers heat inducibility to new host cells, indicating that the conditional function is a property of the viral genome. Unlike previously described temperature-sensitive SV40 mutants, in (ts)-1501 is capable of productive infection in permissive cells at the conditional temperature. The morphology, growth, and oncogenicity of in (ts)-1501-transformed cells at 37 degrees C are similar to those of cell lines transformed by wild-type SV40. HK10-c2(in(ts)-1501), a cloned cell line, transformed at 37 degrees C by the mutant virus, exhibits a transient increase in DNA synthesis before cell death at the conditional temperature. Many properties of in(ts)-1501 are analogous to those of the heat-inducible mutants of bacteriophages in which a heat-inactivated protein is responsible for the stable integration of the prophage in the bacterial chromosome.     

Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus.

We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants. These cell lines were used to recover viruses containing new mutations at the pol locus. Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated. These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells). Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts. Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9. A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.     

Role of vaccinia virus A20R protein in DNA replication: construction and characterization of temperature-sensitive mutants

Previous analyses of randomly generated, temperature-sensitive vaccinia virus mutants led to the mapping of DNA synthesis negative complementation groups to the B1R, D4R, D5R, and E9L genes. Evidence from the yeast two-hybrid system that the D4R and D5R proteins can interact with the A20R protein suggested that A20R was also involved in DNA replication. We found that the A20R gene was transcribed early after infection, consistent with such a role. To investigate the function of the A20R protein, targeted mutations were made by substituting alanines for charged amino acids occurring in 11 different clusters. Four mutants were not isolated, suggesting that they were lethal, two mutants exhibited no temperature sensitivity, two mutants exhibited partial temperature sensitivity, and two mutants formed no plaques or infectious virus at 39 degrees C. The two mutants with stringent phenotypes were further characterized. Temperature shift-up experiments indicated that the crucial period was between 6 and 12 h after infection, making it unlikely that the defect was in virus entry, early gene expression, or a late stage of virus assembly. Similar patterns of metabolically labeled viral early proteins were detected at permissive and nonpermissive temperatures, but one mutant showed an absence of late proteins under the latter conditions. Moreover, no viral DNA synthesis was detected when cells were infected with either stringent mutant at 39 degrees C. The previous yeast two-hybrid analysis together with the present characterization of A20R temperature-sensitive mutants suggested that the A20R, D4R, and D5R proteins are components of a multiprotein DNA replication complex.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.     

DNA-negative temperature-sensitive mutants of herpes simplex virus type 1: patterns of viral DNA synthesis after temperature shift-up.

Temperature-sensitive mutants of herpes simplex virus type 1 belonging to four DNA- complementation groups exhibited two distinct patterns of viral DNA synthesis after shift-up to the nonpermissive temperature. In cultures infected with mutants belonging to complementation groups A, C, and D, little or no viral DNA was synthesized after shift-up. In cultures infected with a mutant in complementation group B, nearly normal amounts of viral DNA were synthesized after shift-up.     

Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1.

A DNA- temperature-sensitive mutant of herpes simplex virus type 1 exhibiting thermolabile DNA polymerase activity, tsD9, was shown to be resistant to phosphonoacetic acid (PAA) when plated at the permissive temperature. ts+ revertants of tsD9 were PAA sensitive and exhibited DNA polymerase activity intermediate between that of the wild-type virus and tsD9, indicating that both temperature sensitivity and sensitivity to PAA are controlled by the same gene. Since the position of tsD9 on the existing herpes simplex virus type 1 linkage map is known, the locus for PAA resistance--and therefore for the structural gene for viral DNA polymerase--has been identified.     

DNA damage and biological expression of adenovirus: a comparison of liquid versus frozen conditions of exposure to gamma rays

Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.     

Preliminary Physiological Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.     

Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation.

Twelve temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1), representing seven complementation groups, were isolated subsequent to 5-bromodeoxyuridine mutagenesis. These mutants were identified by their inability to replicate in a line of monkey (CV-1) cells at 39 C. Seven of these mutants, representing six complementation groups, induced thymidine kinase (tk) and transformed Ltk- cells, a line of mouse L cells lacking tk, to a tk+ phenotype at both the permissive (34 C) and nonpermissive (39 C) temperatures. Thus, the defective cistrons in these six complementation groups, although necessary for lysis, have no essential function in this transformation system. Transformation by these 12 mutants was dependent on prior UV irradiation. Infection of cells with unirradiated virus under conditions which did not permit virus replication was not sufficient to allow cell transformation. Five mutants, representing two complementation groups, were tk- and were incapable of causing the tk--to-tk+ transformation at either 34 C of 39 C. The tk defects in these mutants are probably unrelated to the ts defects, since one of these complementation groups contains a tk+ member. Therefore, transformation of Ltk- cells to a tk+ phenotype by HSV-1 requires an active viral tk gene. One complementation group was represented by a single tk- member. The role of this cistron in transformation remains undetermined since the primary block to transformation is presumed to be the tk- phenotype. Mutants representing the seven complementation groups were unable to replicate at 39 C in two lines of HSV-1-transformed cells, indicating that the activities of resident wild-type copies of the defective cistrons, if present, could not be detected by complementation.     

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.     

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.