Avian sarcoma virus PRCII: Conditional mutants temperature sensitive in the maintenance of fibroblast transformation

Avian sarcoma virus PRCII was mutagenized with 5-azacytidine, and from single foci of transformed cells induced by this virus preparation three conditional mutants were isolated that are temperature sensitive for oncogenic transformation of fibroblasts. Two of these, LA42 and LA47, are not affected in their replication potential at the nonpermissive temperature (41.5°) nor are the virions heat labile. The third mutant, LA46, is coordinately temperature sensitive in replication and transformation. A shift of the incubation temperature from 36° to 41.5° at any time after infection causes transformed cells by any one of the three mutants to revert to normal morphology.     

Temperature-sensitive host-dependent mutants of Sindbis virus

Following chemical mutagenesis of Sindbis virus, two viral clones (clone 35 and clone 58) were isolated which at 34.5° had a relative plaquing efficiency on Aedes albopictus mosquito cells 10⁴- to 10⁵-fold lower than that of wild type virus. Standard growth curves showed that at 34.5° the viral mutants were restricted in mosquito cells but not in primary cultures of chick embryo fibroblasts (CEF). Adsorption of intact virus particles of clone 35 and clone 58 to mosquito cells was as efficient as that observed with wild type virions. However, transfection of mosquito cells with viral RNA of these mutant clones gave a significantly lower plaquing efficiency than infectious RNA from standard virus. Both mutants were temperature sensitive in CEF as well as in A. albopictus cells. At the nonpermissive temperature they were RNA^? and by complementation analysis were assigned to group F. Clones 35 and 58 did not complement each other.     

Immunocytochemical localization of gp70 over virus-related submembranous densities in ts mutant Rauscher murine leukemia virus-infected cells at the nonpermissive temperature.

An indirect immunocytochemical labeling technique employing antibody bound to colloidal gold particles as a marker was used to study the distribution of the major viral glycoprotein gp70 on surfaces of cells and virus in cultures infected with wild-type and mutant ts29 Rauscher murine leukemia virus. The temperature-sensitive mutant-infected cells grown at the nonpermissive temperature acquire submembranous densities which are believed to contain the gag gene-coded uncleaved polypeptide precursor. The cell membranes associated with these densities are heavily labeled with the gold-colloid marker and possess much larger quantities of gp70 than the rest of the cell surface. The budding and extracellular virions produced by these cells after shiftdown to the permissive temperature are also heavily labeled. These findings are consistent with the view that structural protein-membrane-glycoprotein interactions are important in type C virus assembly.     

ts Transformation mutants of avian sarcoma virus PRCII: lack of strict correlation between transforming ability and properties of the P105-associated kinase

Three mutants of avian sarcoma virus PRC-II, LA42, LA46, and LA47, have a temperature-sensitive (ts) lesion affecting cellular transformation in vitro. At the nonpermissive temperature (41.5°) they do not induce focus formation in fibroblast cultures. LA46 also fails to induce colonies in soft agar at 41.5°, while LA42 and LA47 have retained this ability. The mutations appear to be located in the transformation-specific insert of the defective sarcoma virus genomes, since association with different wild-type (wt) helper viruses does not lead to changes in the transforming phenotypes. The transformation-specific protein P105 of PRCII is detectable at the nonpermissive temperature in moderately reduced quantity in wt- and LA42-infected cells, while the amounts of P105 precipitable from LA47-infected cultures under these conditions are significantly decreased. LA46 made barely detectable quantities of P105 at 41.5°. This temperature sensitivity of LA46 in the synthesis of P105 may reflect the greatly reduced levels of transformation-specific RNA in LA46-infected cells at 41.5°. Intracellular phosphorylation of P105 was not found to be ts in the mutants or in wt PRCII at both serine and tyrosine acceptor sites. P105 extracted from wt-, ts mutant- or wt-revertant-infected cells at permissive and nonpermissive temperatures did not vary significantly in the specific activity of its associated protein kinase as assayed in vitro by phosphorylation of P105 itself. However, preincubation of P105 in vitro at 41.5° revealed greater instability of protein kinase reactions measured in P105 immunoprecipitates from mutant- as compared to wt-infected cells. Also the elevation of cellular phosphotyrosine, characteristics of PRCII-transformed cells, was greatly reduced in ts mutant-infected cells at the nonpermissive temperature but was restored to wt levels in genetic revertants derived from the ts mutants. These observations suggest that there is no direct correlation between in vivo or in vitro phosphorylation of P105 and the induction of all parameters of oncogenic transformation. The increase of total cellular phosphotyrosine appears to be correlated with focus formation, but not with the ability to induce agar colonies.     

Phosphorylation of pp60src and the cycloheximide insensitive activation of the pp60src-associated kinase activity of transformation-defective temperature-sensitive mutants of Rous sarcoma virus

Chick embryo fibroblasts infected with two transformation defective temperature-sensitive mutants of Rous sarcoma virus (GI 202 and GI 251), when shifted from the nonpermissive temperature (42°) to the permissive temperature (35°) show a rapid increase in the detectable pp60^src-associated kinase activity. When such cells are shifted back to the nonpermissive temperature there is an equally rapid loss of demonstrable kinase activity. The rapid increase in detectable kinase activity is not substantially hindered by cycloheximide at concentrations inhibiting all de novo protein synthesis, and is associated with a cycloheximide-insensitive phosphorylation of the mutant pp60^src.     

An avian sarcoma virus mutant that is temperature sensitive for virion assembly.

The temperature-sensitive defect in replication of LA334, a double mutant of Rous sarcoma virus, has been characterized both biologically and biochemically. This mutant is complemented for replication at the nonpermissive temperature by both leukosis virus and by RSV(?). Both experiments indicate that LA334 synthesizes functional glycoproteins at the restrictive temperature. This conclusion was confirmed by interference experiments which showed that LA334 induced specific cellular resistance at 41° against superinfection with viruses of subgroup C. Noninfectious virus particles are synthesized at 41° and possess a higher density in sucrose gradients (1.175 g/ml) when compared to those produced at 35° (1.15 g/ml). In addition to the major viral structural proteins these noninfectious virions contain four novel polypeptides, at least three of which appear to be viral in origin. An analysis of viral polypeptide processing indicates that the rate of cleavage of the polyprotein precursor to nonglycosylated structural polypeptides is reduced significantly at the restrictive temperature. Addition of cycloheximide to infected cultures does not prevent the rapid production of infectious virus, seen on shifting cells from the restrictive to the permissive temerature, which suggests that proteins synthesized at 41° can be processed correctly at 35°. Large budding structures that lack the characteristic morphology of C-type particles are visible in electron micrographs of cells infected with the mutant at the nonpermissive temperature. Large virions corresponding in size to some of the budding structures are detectable in negatively stained preparations of noninfectious virus.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) II. Isolation and characterization of additional temperature-sensitive mutants of SV40.

Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive (ts) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the tsA mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for tsBCll previously. Mutants in this group did not produce V antigen at high temperature.     

Electron microscopic studies of temperature-sensitive mutants of herpes simplex virus type 1

Fifteen temperature-sensitive (ts) mutants of herpes simplex virus type 1, each representing a single complementation group, were found to fall into three classes with regard to the degree of defectiveness in virion synthesis which they exhibited at the nonpermissive temperature (39°) when examined by electron microscopy. Mutants in class A, one DNA and one DNA⁺, failed to synthesize detectable particles. Mutants in class B, three DNA^? and four DNA⁺, synthesized small to moderate numbers of empty nucleocapsids. An unique cylindrical core form was observed in particles synthesized after infection with a class B mutant at 39°. Six DNA⁺ mutants in class C synthesized large numbers of virus particles most of which contained apparently empty nucleocapsids. In addition, five of the six mutants synthesized small to moderate numbers of dense-cored nucleocapsids and of these, two formed enveloped, dense-cored particles. The ultrastructural appearance of cells infected with one class C mutant at 39° resembled wild-type virus-infected cells at this temperature, yet it produced 10,000-fold less infectious virus than cells infected with the wild-type virus.     

Palindrome and palindrome-like sequences of herpes simplex virus DNA.

Temperature-sensitive (ts) replication mutants of the Rauscher (R-) strain of murine leukemia virus (MuLV) defective in post-translational processing of the 65,000 molecular weight gag-gene coded precursor (Pr65) have been used to study the initial stages in type-C virus assembly. In is mutant infected cells grown at the nonpermissive but not the permissive temperature, dense material was found below the cell membrane by electron microscopy. Within 30 sec following shiftdown of is mutant infected cells to the permissive temperature (31°) a decrease in the amount of this dense material was observed, coincident with the initiation of virus assembly. Virus assembly and the release of mature virions were completed within 2–4 min. Immunofluorescent studies using monospecific antibodies to several highly purified viral structural proteins revealed a high concentration of these proteins below the cell membrane which also diminished concurrently with virus release. From the evidence presented we propose that the submembranous dense material observed by electron microscopy represents a pool of the polypeptide precursor Pr65, which is cleaved and then incorporated into the developing virus within seconds following shiftdown of the cells to the permissive temperature.     

A mutant of Rous sarcoma virus with a conditional defect in the determinant(s) of viral host range.

A temperature-sensitive mutant of the Prague strain of Rous sarcoma virus of subgroup C, tsPH734PR-C, replicates much less efficiently at the nonpermissive (41°) than the permissive (35°) temperature while transforming equally well at both temperatures. In contrast, the wild-type parent, wtPR-C, is able to replicate and to transform chick embryo fibroblasts equally well at both 35° and 41°. Two lines of evidence suggest that tsPH734PR-C is defective in the synthesis or utilization of virus envelope glycoproteins GP85 and GP35. First, tsPH734PR-C appears to be defective in the incorporation of the virus envelope glycoproteins GP85 and GP35 into the noninfectious virus particles synthesized at 41°. Second, tsPH734PR-C, a host range subgroup C virus, is not complemented for replication of subgroup C virus at 41° by coinfection of cells with RAV-6 avian leukosis virus of subgroup B or by coinfection with the defective Bryan strain of Rous sarcoma virus, BH-RSV(?).     

Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus

Methods have been developed for rapid isolation and genetic analysis of vaccinia virus mutants. These methods include: (1) monitoring mutagenesis by measuring conversion of wild-type, phosphonoacetic acid-sensitive virus to phosphonoacetic acid-resistant virus, (2) screening for mutants by plaque enlargement, and (3) a qualitative spot test for complementation. Twenty-six temperature-sensitive mutants of vaccinia virus have been isolated. All have reversion indices of 10^?4 or less. One-step growth experiments have been done at 40° and 31° with all the mutants and in all cases the virus yield at 40° is less than 8% of the yield observed at 31°. Complementation analysis has been completed on all 26 mutants, showing that these mutants together comprise 16 complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize viral DNA at the nonpermissive temperature. The results show that 3 of the 24 have a DNA-negative phenotype. These three mutants fall into two complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize early and late viral proteins at the nonpermissive temperature. From this analysis, four phenotypes appear: (1) normal, (2) a phenotype associated with DNA-negative mutants characterized by prolonged synthesis of early proteins and the absence of late protein synthesis, (3) weak or slow late protein synthesis, (4) abortive late protein synthesis.     

Pathway of assembly of herpesvirus capsids: An analysis using DNA+ temperature-sensitive mutants of pseudorabies virus

It was reported previously that nine different DNA⁺ is mutants of Pr virus were all defective in the processes leading to the cleavage of concatemeric viral DNA and to the formation of nucleocapsids. Six of these mutants were found to assemble capsids at the nonpermissive temperature; three were capsid^? (Ladin et al., 1980). In the present communication we have characterized these mutants further. Each of the mutants was rescued by one specific restriction fragment of wild type virus DNA indicating that none of these mutants is altered in more than one locus. Many of the mutants complemented one another well at a frequency indicating that they are probably mutated in different genes. Cells infected at the nonpermissive temperature (41°) with the DNA⁺ mutants synthesized all the virus proteins synthesized by wild type-infected cells (as detected by PAGE) with two notable exceptions: (1) ts1-infected cells synthesized a thermolabile 142K capsid protein. (2) In ts1, ts109, and tsJ (all capsid^? mutants)-infected cells, detectable amounts of 35K capsid protein did not accumulate. The 35 K capsid protein is normally processed from a precursor protein, the processed protein being detectable in the nucleus of the infected cells only. In capsid^?-infected cells processing of the 35K protein precursor did not occur. Furthermore, accumulation in the nucleus of all capsid proteins was impaired in all the capsid^?-infected cells. Because at least two of the three capsid^? mutants are mutated in different genes, we conclude that the processing of the 35K protein, capsid assembly, and accumulation of capsid proteins in the nuclei are interdependent events which occur only if all the proteins necessary for capsid assembly are functional. On the basis of these results, we propose that the 35K protein is processed during capsid assembly and that assembly of the proteins into capsids is required for the continued movement of capsid proteins to, and their accumulation in, the nuclei of infected cells.