Priscilla A. Schaffer

Temperature-sensitive mutants of herpes simplex virus type 1: Isolation, complementation and partial characterization

Abstract Twenty-two temperature-sensitive mutants of herpes simplex virus type 1 isolated following mutagenesis with 5-bromodeoxyuridine, nitrosoguanidine, and ultraviolet light were classified according to their ability to complement each other at 39°, the nonpermissive temperature. Fifteen nonoverlapping complementation groups have been identified. Partial biochemical characterization of mutants has demonstrated that members of 4 groups were unable to synthesize viral DNA at 39°. Several mutants were found to differ from the wild-type virus with regard to thermal stability at 39°, suggesting that they may possess altered structural proteins.

Recombination between temperature-sensitive mutants of herpes simplex virus type 1☆

Abstract Fifteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 representing 10 complementation groups were examined for their ability to recombine. Efficient recombination was demonstrated between most mutant pairs in standard two-factor crosses. Mutants which failed to complement each other also failed to recombine or recombined with low frequency. Progeny analysis of putative ts+ recombinants demonstrated that complementing clumps and/or multiploid particles did not contribute significantly to recombinant yields. Using recombination data from crosses between 11 mutants representing 7 complementation groups, a provisional linkage map has been constructed. The map spans approximately 38 recombination units and is linear.

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

Abstract 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.

A temperature-sensitive mutant of herpes simplex virus defective in glycoprotein synthesis☆

Abstract A temperature-sensitive mutant of herpes simplex virus (HSV) type 1, ts343, has been isolated from 5-bromodeoxyuridine-treated and HSV-infected human embryonic lung (HEL) cell cultures. The mutant failed to replicate at the nonpermissive temperature (40°) whereas the virus yield at the permissive temperature (35°) was about 5 × 106 plaque-forming units/ml. The temperature-sensitive defect of ts343 was not due to increased thermal lability of the virus or to its failure to adsorb to HEL cells. Temperature shift-up experiments showed cessation of virus synthesis followed by a drop in titer, while shift-down experiments showed reversibility of the temperature-sensitive defect. Viral DNA synthesis was not detected when measured by CsCl equilibrium centrifugation of lysates of thymidine-3H-labeled ts343-infected cells incubated at the nonpermissive temperature. Virus-specific antigen synthesis, to a limited degree, occurred at this temperature. Electron microscopic examination of thin sections of cells infected with ts343 and maintained at 40° revealed a limited number of intranuclear naked viral capsids but the complete absence of enveloped particles. Polyacrylamide gel electrophoresis was employed to investigate possible differences in polypeptide and glycopeptide synthesis induced by ts343 and the wild-type virus. Coelectrophoresis of wild-type and ts343 virus-induced cytoplasmic proteins synthesized at 35° exhibited identical electrophoretic patterns. However, at 40° the electrophoretic profile differed significantly with respect to a nearly complete absence of protein C5, identified as the major envelope protein. The electrophoretic profile of the glucosamine-3H-labeled fraction of ts343-infected cells demonstrated that protein C5 was the major glycosylated protein synthesized at 35° within the cytoplasm, but at 40° glycoproteins were not made. By contrast, glycoprotein synthesis by wild-type virus was not inhibited at 40°. The kinetics of glucosamine incorporation by ts343 at 35° was maximal between 6.5 and 10.5 hr post infection, whereas at 40° the uptake of glucosamine into TCA-precipitable material decreased throughout the 12 hr of observation.

Isolation, complementation and preliminary phenotypic characterization of temperature-sensitive mutants of herpes simplex virus type 2.

Abstract Eight temperature-sensitive ( ts ) mutants of herpes simplex virus type 2, isolated following mutagenesis with 5-bromodeoxyuridine, were assigned to seven nonover-lapping complementation groups (A through G). Partial phenotypic characterization of the mutants at the nonpermissive temperature (38 °) revealed the following: (1) Mutants of 3 complementation groups had a DNA − phenotype, the remaining being DNA + . (2) The DNA − mutants were deficient in DNA polymerase activity, whereas the DNA + mutants were not. (3) Two mutants, one DNA + and one DNA − , failed to induce thymidine kinase. (4) Except for one DNA − mutant, all mutants synthesized physical particles; however, only DNA + mutants synthesized enveloped particles. (5) Mutants belonging to 4 complementation groups were markedly more thermolabile than the wild-type virus, suggesting that they possessed alterations in viral structural proteins.

Thymidine kinase activity of herpes simplex virus temperature-sensitive mutants.

Eighteen temperature-sensitive ( ts ) mutants of herpes simplex virus type 1 were studied with regard to their ability to synthesize virus-induced thymidine kinase (TK) at permissive

Intertypic complementation and recombination between temperature-sensitive mutants of herpes simplex virus types 1 and 2.

Abstract In an attempt to identify functionally “common” and “type-specific” genes in the genomes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), intertypic complementation and recombination experiments between temperature-sensitive (ts) mutants of these viruses were undertaken. Intertypic complementation was carried out with seven HSV-1 ts mutants representing five complementation groups, and eight HSV-2 ts mutants representing five complementation groups. Efficient complementation was detected with most HSV-1 + HSV-2 ts mutant pairs, indicating extensive interchangeability of gene functions between HSV types 1 and 2. Progeny analysis of complementation yields demonstrated that complementation was symmetric; i.e., equal amounts of both HSV-1 and HSV-2 ts mutants were produced. HSV-1 wild-type virus was also able to complement HSV-2 ts mutants efficiently. Intertypic recombination experiments demonstrated that heterologous mutant pairs which failed to complement also failed to recombine efficiently.

Enhanced Replication of Temperature-Sensitive Mutants of Herpes Simplex Virus Type 2 (HSV-2) at the Nonpermissive Temperature in Cells Transformed by HSV-2

In order to determine whether herpes simplex virus type 2 (HSV-2) genetic information present within HSV-2-transformed cells could assist the replication of temperature-sensitive (ts) mutants of HSV-2 at the nonpermissive temperature (38°), the replication of 8 ts mutants of HSV-2 was examined in normal, SV40-transformed, and HSV-2-transformed hamster cells at 38°. The growth of two DNA-positive ts mutants was significantly enhanced in two HSV-2-transformed cell lines at 38° as compared with their growth in normal cells. No enhancement of growth of any of the eight ts mutants occurred in SV40-induced tumor cells. These results suggest that resident, functional HSV genetic information in HSV-2-transformed cells assisted the replication of the mutants by complementation mechanisms.

Complementation of adeno-associated satellite viral antigens and infectious DNA by temperature-sensitive mutants of herpes simplex virus

Abstract Production of type 4 adeno-associated satellite virus (ASV) structural antigens and infectious DNA was studied using temperature-sensitive ( ts ) mutants of herpes simplex virus (HSV) types 1 and 2. Seven of the eight mutants studied were unable to synthesize HSV DNA at the nonpermissive temperature. Mutants belonging to three HSV-1 and two HSV-2 complementation groups complemented ASV antigen production while members of two other HSV-1 groups did not. Infectious satellite virus DNA was isolated from the HSV-1 ts -1 complementing system.

Marker rescue of temperature-sensitive mutants by defective DNA of herpes simplex virus type I

Abstract High-density, defective DNA ( ϱ CsCl = 1.732 g/cm 3 ) produced in infections of the 15th through 20th undiluted serial passage of herpes simplex virus type 1 (HSV-1), Strain KOS, was purified by preparative CsCl density gradient ultracentrifugation. Purified defective DNA expressed no discernible cytopathic effects when transfected into receptive cell monolayers. Cleavage of defective DNA preparations with EcoRI restriction endonuclease revealed four major and three minor bands upon electrophoresis in agarose gels. Restriction endonuclease Hind III failed to cleave defective DNA. Marker rescue studies revealed that of 13 temperature-sensitive mutants representing 12 complementation groups, only mutants in group B were rescued by defective DNA. These studies demonstrate, therefore, that defective DNA is enriched for at least part of the B cistron and suggest that this cistron is located in the region of the viral genome from which defective DNA is generated.