Morphological phenotype of temperature-sensitive mutants of simian virus 40 in productive infection.

Temperature-sensitive mutants of simian virus 40 from the complementation groups I (viral-DNA positive and V-antigen positive), II (viral-DNA positive and V-antigen negative), and III (viral-DNA negative and V-antigen negative) were examined by electron microscopy for their ability to synthesize physical virus particles at the nonpermissive temperature (40°) in productive infection. Examinations of thin sections of infected cells revealed that all the group I mutants studied were not temperature-sensitive for the synthesis of physical virus particles in terms of the proportion of cells synthesizing virions, approximate number and mode of arrangement of virions in the virus-producing cells, and morphology of virions present in the cells. Representative mutants of the complementation groups II and III were temperature-sensitive for the synthesis of virions or virion-related structures. The virus particles of the group I mutants produced at 40°, in contrast to those produced at the permissive temperature (33°), were readily destroyed after lysing the cells by repeated freezing and thawing or by sonication, but the lysate still retained V antigen, the amount of which was similar to that in lysates from the mutant-infected cells at 33° and from wild-type-infected cells at 33 and 40°. Temperature-shift experiments suggested that: (1) The event that is temperature sensitive in a group I mutant infection occurs about the time of the appearance of new infectious virions, suggesting that the mutation affects the final stage of virus maturation; (2) the affected function of the group II mutant occurs at about 10 hr prior to the time of the appearance of new infectious virions at 33°, although the mutant employed may be regarded as a late mutant because of the heat lability of the virions produced at 33° and because viral DNA synthesis is normal at 40°; and (3) the group III mutant temperature presented suggest that the three genetically distinct groups of mutants represent three functionally distinct processes in the replicative cycle of simian virus 40.     

The fine structure of cells infected with temperature-sensitive mutants of herpes simplex virus type 2.

The fine structure of cells infected with the HG 52 strain of herpes simplex virus type 2 and 13 temperature-sensitive mutants derived from it was investigated. In cells infected with the wild-type virus, development of virions appeared to be similar to that described in previous reports. However there were two exceptions to this: (1) capsid envelopment apparently occurred de novo in the nucleus; (2) densely staining vacuolar accumulations were seen, frequently surrounding virus capsids. The 13 temperature-sensitive mutants of the virus were divided into three classes according to the type of capsid, if any, produced in cells infected and maintained at the non-permissive temperature. Class I mutants produced no capsids, Class II mutants produced empty and partial-cored capsids and Class III mutants produced empty, partial- and dense-cored capsids. Cellular alterations were also determined. Membranous tubular structures, previously unreported for herpes simplex virus, were observed in cells infected with Class III mutants and very occasionally with wild-type virus at the non-permissive temperature. Cytoplasmic particles were also found, but could not be correlated with any particular class of mutant.     

Genetic and phenotypic characterization of mutants in four essential genes that map to the left half of HSV-1 UL DNA

Several HSV-1 proteins including the major capsid protein (VP5), two minor capsid proteins (VP11-12 and VP18.8), the alkaline nuclease and glycoprotein gH have been reported to be encoded by the left-most one-third of HSV-1 UL DNA. In this paper, we present physical mapping data and phenotypic analysis of six ts mutants whose mutations lie within this region and which collectively represent four functional complementation groups (1-6, 1-7, 1-10, and 1-26). In this study, mutants in complementation group 1-10 were found to be defective in the synthesis of viral DNA, late viral polypeptides, and the formation of mature capsid-like structures--properties characteristic of other ts mutants defective in functions required for viral DNA synthesis. Two DNA-positive mutants in complementation group 1-7 fail to induce capsid formation and probably possess mutations in coding sequences for VP5. Mutants in two other complementation groups (1-6 and 1-26) synthesize significant levels of viral DNA, late polypeptides, and capsids. The functions of the gene products represented by these mutants remain to be determined.     

Three complementation subgroups in group IV RNA phage SP

We isolated twelve suppressor sensitive (sus) mutants from the RNA phage SP which belongs to group IV and classified them into three cistrons (genes 1, 2, and 3) by complementation tests. Using representative mutants of each cistron, synthesis of infectious RNA, phage antigen (serum blocking power) and defective particles under nonpermissive conditions were examined. Gene 1 mutants had a defect in maturation protein synthesis, gene 2 mutants in coat protein synthesis, and gene 3 mutants in RNA synthesis, respectively. Thus, group IV RNA phage SP has (at least) three genes corresponding to RNA replicase, coat protein, and maturation protein, as already shown in group I (f2, R17) and group III (Qβ) RNA phages.From intergroup complementation tests between several sus mutants of groups I, III, and IV, Qβ(III) had a fairly close relationship to SP(IV), while f2(I) had no such close relationships to Qβ(III) or SP(IV). These are consistent with the results of grouping by several biological and physicochemical criteria and support the idea that groups I and II may be assembled into one group and groups III and IV into another.     

The selection of wild-type revertants from methotrexate permeability mutants

In a previous report, we described the selection and partial characterization of three distinct classes of methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained a structural alteration in dihydrofolate reductase. Class II cells showed an alteration affecting the permeability of the drug. Class III cells, selected from class I cells, had an increased activity of the altered enzyme. In the work described here, the sensitivity of these lines to the diaminopyrimidines has been investigated. Class I cells are as sensitive, class II cells are 5- to 10-fold more sensitive, and class III cells are 10- to 30-fold more resistant than wild-type cells. The increased sensitivity of the class II cells provided an opportunity to select for revenants of these mutants and such phentotypic wild-type revenant cells have been selected using one diaminopyrimidine, pyrimethamine. Such cells have drug sensitivities and permeability characteristics similar to wild-type cells. A second class has been identified which has wild-type drug sensitivities to the diaminopyrimidine s but Mtx class II resistance to Mtx, and drug permeabilities characteristic of Mtx-resistant class II cells.     

Biochemical characteristics of fowl plague virus TS mutants

Five fowl plague virus ts mutants belonging to five different complementation groups were studied. In one mutant (ts 131) the virion RNA-polymerase was defective in vitro at the nonpermissive temperature and the later stages of replication in vivo (synthesis of RNA, polypeptides, and RNP) were found to be defective. One of the mutants studied (ts 5) under nonpermissive conditions showed no disorders in the function of virion RNA-polymerase or in vivo synthesis of RNA, polypeptides, functionally active hemagglutinin and neuraminidase or RNP production, however, no formation of infectious virions or virion-like structures occurred under these conditions. In three mutants (ts 43, ts 166, and ts29) the virion RNA-polymerase was capable of functioning in vitro at the nonpermissive temperature but no RNA synthesis in vivo was observed when tested 4 hr after infection. In these mutants, synthesis of virus-specific polypeptides at nonpermissive and optimal temperatures under the conditions of the experiment was similar both qualitatively and quantitatively. Despite the fact that in the cells infected with ts 43, ts 166, and ts 29 mutants synthesis of both polypeptides hemagglutinin precursors was observed at the nonpermissive temperature, no functionally active hemagglutinin was produced under these conditions. The data are discussed with reference to the mechanisms of reproduction of orthomyxoviruses.     

Complementation between temperature-sensitive (ts) and host range nontransforming (hr-t) mutants of polyoma virus.

Two major classes of polyoma mutants are defective in cell transformation: early temperature-sensitive mutants of the tsA type which are defective in viral DNA synthesis and transformation at 39°, but not at 32°; and host range nontransforming (hr-t) mutants which fail to transform at either temperature. Mixed infection of mouse 3T3 cells by hr-t mutants and early tsA-type mutants results in enhanced growth of the tsA-type mutants at 39°, indicating that the hr-t mutants can supply the early viral function required for viral DNA synthesis. The hr-t mutants also complement late is mutants which fail to produce infectious progeny at 39° because of alterations in the 45,000-dalton major virion protein. Mixed infection of hamster BHK or rat Y1 cells by hr-t and tsA-type mutants results in efficient transformation at 39°, indicating that the two classes of mutants can complement for transformation. No complementation is observed in pair-wise crosses among the early tsA-type mutants alone. The tsA-type mutants are located in the distal portion of the early region of the polyoma genome [Miller, L. K., and Fried, M. (1976) J. Virol.18, 824–832]. The hr-t mutants are located in the proximal portion [Feunteun, J., Sompayrac, L., Fluck, M., and Benjamin, T. (1976) Proc. Nat. Acad. Sci. USA]. These results suggest that the early region of the polyoma genome is divided into two functional regions which can complement for transformation. The ts3 mutant of polyoma is located in the proximal portion of the late region.     

Three new complementation groups of temperature-sensitive Chinese hamster ovary cell mutants defective in the endocytic pathway

We describe here the results of complementation studies with six mutant Chinese hamster ovary cells expressing temperature-sensitive lesions affecting the endocytic pathway. The mutants were crossed with representatives of the End1 and End2 complementation groups identified previously by Robbins et al. (J. Cell Biol. 99:1296–1308, 1984). Two mutants, G.8.1 and 31.1, were members of the End1 complementation group. One mutant, 25.2, was a member of the End2 complementation group. The other three mutants each defined new complementation groups, which we have designated End3 (mutant G.7.1), End4 (mutant V.24.1), and End5 (mutant 42.2). Previous work on mutants of the Endl, End2, and End3 classes had shown that these mutants were defective in endosomal acidification. We prepared postnuclear supernatants from mutants harvested at the nonpermissive temperature and compared their acidification activities, assessed by ATP-stimulated quenching of acridine orange. Members of the End1, End2, and End2 groups had reduced acidification activity, correlating with the acidification defects known to be expressed by these mutants. Strain V.24.1 (End4) also expressed a 40% reduction in acidification activity, while strain 42.2 (End5) had no reduction of acidification activity.     

Infection of human cells with polyoma virus

Human cells which are resistant to infection with polyoma virus (PV) and PV-DNA by the conventional virus-absorption method were infected by microinjection of the virus or the viral-DNA into the cell cytoplasm. Induction of PV-tumor (T) antigen production and synthesis of cellular DNA were found in a large number of the infected cells, but no viral capsid (V) antigen was detected. Among 3 × 10³ virus-injected cells, none developed into a transformed cell colony. Multinucleated human muscle cells (myotubes), in which DNA synthesis and mitosis are irreversibly repressed after microinjection of PV, do not incorporate [³H]thymidine but produce T-antigen.     

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.     

Further isolation and characterization of temperature-sensitive mutants of influenza virus.

We have assigned 34 temperature-sensitive (ts) mutants of the WSN strain of influenza virus into seven nonoverlapping recombination-complementation groups by conducting pairwise crosses. A single gene of the viral genome was affected in 27 mutants while six were double mutants and one was a triple mutant. The synthesis of virion RNA in mutant-infected cells at the nonpermissive temperature was studied by the incorporation of [³H]uridine in the presence of actinomycin D into an acid-insoluble product. This method separated mutants into two classes. Mutants belonging to Groups I, II, III, and V were RNA^?, while those belonging to Groups IV, VI, and VII were RNA⁺. The results of temperature shift-up experiments suggested that all four groups of RNA^? mutants were defective in replicating virion RNA. Production of serologically or functionally active subviral components (ribonucleoprotein, hemagglutinin, and neuraminidase) by RNA^? mutants at the nonpermissive temperature was variable. RNA⁺ mutants produced a normal amount of these components, except Group IV mutants in which the production of functional hemagglutinin and neuraminidase was greatly depressed.     

Six complementation classes of conditionally lethal protein synthesis mutants of CHO cells selected by3H-amino acid

Using a tritiated amino acid suicide procedure designed specifically to select conditional protein synthesis mutants, we have isolated and characterized a large number of such mutants of Chinese hamster ovary cells. All of the mutants are genetically stable and behave as recessives in somatic cell hybrids. Most of the new mutants are phenotypically dependent on the concentration of a specific amino acid as well as on temperature. In addition to identifying many additional leucyl- and asparagyl-tRNA synthetase mutants, complementation analysis has distinguished four new genetic classes representing methionine-, glutamine-, histidine-, and arginine-dependent mutants. Biochemical characterization of representative mutants from each of these six classes has identified the primary lesions as being defective aminoacyl-tRNA synthetases. Our selection results further demonstrate the high specificity of the³H-amino acid procedure for isolating protein synthesis mutants. Reconstruction experiments performed with two representative mutants indicated a selection efficiency of approximately 10% under standard conditions.     

Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17.

The polypeptides induced in cells infected with a Glasgow isolate of HSV-I (17 syn+) have been characterized by SDS polyacrylamide gel electrophoresis. Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells. The different polypeptide patterns obtained at permissive (31 degrees C) and nonpermissive (38 degrees C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA -ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles. A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (I) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for different polypeptides. (6) The inhibition of host protein synthesis by the virus may not be a simple single step process.     

Complementation between temperature-sensitive mutants isolated from Aedes albopictus cells persistently infected with Western equine encephalitis virus.

To obtain temperature-sensitive (ts) mutants, Aedes albopictus cells were persistently infected with wild-type Western equine encephalitis virus. Many leaky and a few stable ts mutants were found among small plaque-forming progeny virus from the cultures at the early stage (up to 30 days) after initial infection. At later times (80–170 days) the persistently infected cultures gave rise to small plaque variants with stable ts mutations. Twenty-four stable ts isolates from early and late passages were classified into three groups by RNA phenotype and complementation. They consisted of three RNA⁺ and one RNA^? mutants with single-site mutation from the early cultures. The remaining 20 ts mutants isolated from the late cultures all had multiple-site mutations; 19 of these were double mutants and may be identical, and one was a triple mutant. We also demonstrated positive complementation between single and multiple mutants. All ts mutants isolated from persistently infected cells possess either the group III RNA^? mutation or the group IV RNA⁺ lesion or the double mutation of group III/IV. The defect in the structural protein is likely to be in one of the envelope proteins, the E2 protein. It is possible that a control mechanism that favors the accumulation of such multiple mutants exists in persistently infected mosquito cells.     

Defective transforming capacity of adenovirus type 5 host-range mutants

Host-range (hr) mutants of human adenovirus type 5 (Ad 5), selected for their ability to grow preferentially on transformed human embryo kidney cells (293 cells) and shown to fall into two complementation groups (I and II), have been tested for ability to transform rat embryo, rat embryo brain, and baby rat kidney cells. When embryo or embryo brain cells were used, mutants of both complementation groups either failed to transform or transformed at a frequency much lower than that observed with wild-type (wt) virus. Moreover, the small number of transformed colonies which did arise in hr-infected cultures failed to grow when isolated and subcultured, unlike wt transformants from which lines could be established with a high success rate. When assays were carried out with baby rat kidney cells,hr mutants of complementation group II were again transformation negative, while mutants of group I induced transformation with several times the efficiency of wt Ad 5. Again, attempts to establish transformed lines from single hr-transformed foci were consistently unsuccessful, in contrast to an almost 100% success rate with wild-type transformants; hr mutant-transformed lines could only be established with difficulty by passaging whole cultures. Our results are consistent with the idea that group I mutants are able to initiate an abortive or abnormal transformation but are defective in some aspects of its maintenance; group II mutants, on the other hand, appear to be defective in initiation of transformation.     

Isolation and characterization of mutants of bacteriophage T4 resistant to folate analogs

The dihydrofolate (FH₂) reductase specified by the T4 frd gene (previously called the wh gene) is apparently nonessential for phage growth on Escherichia coli because the bacterial FH₂ reductase can partially substitute for the phage enzyme. To study the effect in vivo of folate analogs which specifically inhibit the T4 FH₂ reductase, the enzyme must be made essential for phage production. Partial inhibition of the E. coli FH₂ reductase with the folate analog trimethoprim strongly inhibits phage production by a T4 frd mutant and slightly inhibits wild-type phage production. Adding an additional T4-specific folate analog, a chlorophenyl triazine, strongly inhibits wild-type phage production without preventing the uninfected E. coli cells from multiplying.We have isolated spontaneous mutants of T4 which are capable of producing progeny in the presence of chlorophenyl triazine and another folate analog pyrimethamine. These mutants are designated folate analog resistant (far) and have been separated into two general classes. Class I mutants induce T4-specific FH₂ reductases which are less sensitive to the action of the folate analogs than the normal FH₂ reductase. Class II mutants induce an unaltered FH₂ reductase but contain mutations in a variety of T4 genes. Some class II mutants induce more phage FH₂ reductase activity than wild-type T4 and appear to be regulatory mutants.