Matilda Benyesh-Melnick

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.

Effects of 2-deoxy-d-glucose on herpes simplex virus replication☆

Abstract The replication of herpes simplex virus (HSV) was studied in BSC1 cells cultured in the presence of various concentrations of 2-deoxy- d -glucose. At all concentrations tested, the yield of infectious virus was inhibited to a greater degree than the yield of physical particles, while the percentage of the total particles which were enveloped remained constant. Analysis of HSV-induced glycoproteins on SDS-polyacrylamide gels (SDS-PAGE) indicated that as the concentration of 2-deoxy- d -glucose increased, there was a decrease in the apparent molecular weights of the viral glycoproteins. Analysis on SDS-PAGE of the viral-induced proteins indicated no significant alterations with the exception of an additional component detectable only in HSV-infected cultures containing the higher concentrations of 2-deoxy- d -glucose. Preliminary SDS-PAGE analysis indicated that 2-deoxy- d -glucose is incorporated into the HSV-induced glycoproteins.

Isolation and characterization of a large molecular-weight polypeptide of herpes simplex virus type 1

Abstract Infection of human embryonic lung cells at the nonpermissive temperature (39°) with tsB2, a DNA-negative mutant of herpes simplex virus type 1 (HSV-1) resulted in the accumulation of a large molecular-weight (175,000) virus-induced polypeptide detectable by SDS-polyacrylamide gel electrophoresis. This polypeptide (VP175) was detectable only in small amounts and was found mainly in the nuclear fraction of cells infected with tsB2 at the permissive temperature (34°) or with wild-type HSV-1 at both 34° and 39°. However at 39° VP175 accumulated to become the major component in both the cytoplasmic and nuclear fractions of tsB2-infected cells. Identical results were obtained with a second mutant (tsB21) in the same complementation group. Temperature-shift studies suggest that the events responsible for the accumulation of VP175 at 39° occur early in the replicative cycle. With the use of a combination of SDS-preparative and analytical disc gel electrophoresis, VP175 was isolated and rabbit antisera to this polypeptide were prepared. By immunofluorescence assay, anti-VP175 sera reacted only with the nucleus of wild-type HSV-1-infected cells, whereas tsB2-infected cells cultured at 39° showed both cytoplasmic and nuclear immunofluorescence. In addition, the anti-VP175 serum reacted preferentially with HSV-1 as compared with HSV-2-infected cells, suggesting a possible type specificity for the reagent.

Physicochemical properties of the DNA of herpes viruses.

Abstract The biochemical and biophysical properties of the DNA of herpes simplex virus type 1 (herpes virus-1) and type 2 (herpes virus-2), infectious bovine rhinotracheitis and pseudorabies viruses were compared. Buoyant densities in CsCl were 1.725, 1.727, 1.730 and 1.731 g/cm 3 for the DNAs of herpes virus-1, -2, infectious bovine rhinotracheitis virus and pseudorabies virus, respectively. Base composition analysis was conducted by enzymatically hydrolyzing viral DNA and calculating the nucleotide composition from elution patterns obtained from anion-exchange column. From these patterns, the guanine plus cytosine content was calculated to be 69.9% for herpes virus-1 DNA, 72.0% for herpes virus-2 DNA, and 76.7% for pseudorabies virus DNA. When thermally denatured in 0.015 M NaCl-0.0015 M sodium citrate, the DNAs of herpes virus-1, -2, infectious bovine rhinotracheitis virus and pseudorabies virus yielded average T m values of 82.6°, 82.7°, 85.6° and 86.6°, respectively. The DNAs of herpes virus-1 and -2 had the same sedimentation coefficient (56 S) in neutral Tris-saline buffer, corresponding to a molecular weight of 88·10 6 . Pseudorabies virus DNA sedimented as two components with sedimentation coefficient values of 26 S and 31 S.

Defective Virions of Herpes Simplex Viruses

Serial undiluted passage of clonally purified herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in 4 different types of cells resulted in (i) partial loss (84–98%) of infectivity and (ii) the app

Studies on the relatedness of herpesviruses through DNA-DNA hybridization

The relatedness of the DNAs of three different herpesviruses, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and pseudorabies (Pr) virus was compared by DNA-DNA hybridization on nitrocellulose membrane filters. Reciprocal and competition hybridization experiments performed under saturation conditions demonstrated a maximum homology of 70% between the DNAs of HSV-1 and HSV-2. Under similar conditions, Pr virus DNA had 8–10% complementarity with either HSV-1 or HSV-2 DNA.

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.

Antigens specified by herpesviruses. I. Effect of arginine deprivation on antigen synthesis.

Abstract The synthesis of herpes simplex virus antigens was studied in BHK21 cells deprived of arginine for 48 hours prior to infection and maintained in the absence of arginine thereafter. Even though infectious virus yields were 10,000-fold lower than those obtained in the presence of arginine, immunofluorescent (IF) and complement-fixing (CF) antigens were detected. However, in the absence of arginine both IF and CF antigens were found primarily in the cytoplasm of the infected cells. Release of the arginine block at 10 hours after infection resulted in the prompt appearance of increasing amounts of nuclear IF and CF antigens; within 6 hours after arginine addition, the extent of nuclear immunofluorescence and the titers of CF antigen in the nuclear fractions approximated those of infected cells that had been maintained in the presence of arginine throughout the 16–18-hour period.

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.

DNA polymerase induction by DNA-negative temperature-sensitive mutants of herpes simplex virus type 2

Abstract Four DNA − temperature-sensitive mutants of herpes simplex virus type 2 (HSV-2), belonging to three complementation groups, were examined for their ability to induce virus-specific DNA polymerase activity in HEp-2 cells. At the permissive temperature (34°), all mutants induced DNA polymerase activity at levels comparable to wild-type HSV-2. At the nonpermissive temperature (38°), all four mutants were deficient in polymerase induction, producing from 0 to 30% of wild-type levels, and this deficiency was not due to a diffusible inhibitor. Although the polymerase of all four mutants was only slightly more heat labile than that of wild-type virus in vitro , two mutants had temperature-sensitive DNA polymerase activity when tested in temperature shift-up experiments. Temperature shift-down experiments established that the proteins responsible for the block in DNA polymerase induction began to function at about 4 hr after infection and were required continuously thereafter. The data obtained suggest that at least three genes of HSV-2 code for polypeptides which either are a part of the virus-specific polymerase or are necessary for its induction and/or regulation