Richard J. Courtney

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.

Polypeptides synthesized in herpes simplex virus type 2-infected HEp-2 cells

Abstract The proteins induced by herpes simplex virus type 2 in infected HEp-2 cells were studied by high-resolution polyacrylamide slab-gel electrophoresis. A total of 51 infected cell-specific polypeptides (ICSP) were detected, accounting for three-fourths of the coding capacity of the virus. The induced polypeptides were allocated to three groups based on their time of synthesis in the virus growth cycle. Cycloheximide treatment of infected cells during the early stage of virus growth was found to cause irreversible inhibition of protein synthesis. Herpes simplex virus type 2 polypeptides which underwent considerable posttranslational modification were detected.

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.

Herpesvirus-induced antigens in squamous-cell carcinoma in situ of the vulva.

Antigens induced by herpes simplex virus Type 2 (HSV2) were found to be associated with squamous-cell carcinoma in situ of the vulva in nine of 10 patients. The HSV2-induced antigens are DNA-binding proteins that are normally present in the nuclei of infected cells, but in the cells of the carcinomas in situ they were found in the cytoplasm. Whole-virion structural antigens were not present, although there was serologic evidence of previous HSV2 infection in patients tested for the presence of antibodies. The observations reported here and the recent parallel rise in the prevalence of both HSV2 infections and vulvar carcinoma in situ, particularly in women under 40 years of age, suggest an association of HSV2 infection with this type of neoplasia, the nature of which remains to be determined.

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.

Cyclic Appearance of Defective Interfering Particles of Herpes Simplex Virus and the Concomitant Accumulation of Early Polypeptide VP175

Serial passage of undiluted herpes simplex virus types 1 and 2 resulted in cyclic production of infectious and defective virions. Defective virus production was characterized by the appearance of a new species of viral DNA with a higher bouyant density in CsCl than standard viral DNA. Measurement of the infectivity titer and DNA synthesis revealed that the defective particles interfered with the replication of standard virions and stimulated the overproduction of a large molecular weight (175,000 daltons) polypeptide.

The synthesis of herpes simplex virus proteins in the absence of virus DNA synthesis

Summary When herpes simplex virus infected cells were blocked from synthesizing DNA, alterations in the production of certain classes of herpes simplex virus specific proteins were detected. Some proteins were made in excess, some in normal amounts, some in reduced amounts and some were not made. An example of each type was chosen, and its kinetics of synthesis studied. Possible explanations of the phenomena are discussed.

Herpes simplex virus protein synthesis in the presence of 2-deoxy-d-glucose

Abstract The proteins and glycoproteins induced by herpes simplex virus type 1 (HSV-1) were labeled with [ 14 C]amino acids or [ 14 C]glucosamine in the presence or absence of 2-deoxy- d -glucose (deoxyglucose) and analyzed by slab gel electrophoresis. In the presence of 0.1% deoxyglucose (6.1 m M ), the major envelope glycoprotein (VP123, MW 123,000) labeled with [ 14 C]glucosamine was shifted to a component of an apparent lower molecular weight (VP123′). In the presence of increasing concentrations of deoxyglucose, there was a progressive decrease in the amount of [ 14 C]amino acids incorporated into polypeptides which normally band in the VP123 region. Concomitant with this decrease was an increase in [ 14 C]amino acids incorporated into a polypeptide(s) of greater electrophoretic mobility and of an apparently lower molecular weight. The polypeptide(s) was designated DG92 (MW 92,000) and was found to be predominantly associated with the nuclear fraction of HSV-1-infected cells cultured in the presence of deoxyglucose. The effects of deoxyglucose on HSV-1 polypeptide synthesis could be prevented by the addition of mannose.

Effects of cytochalasin B on herpes simplex virus type 1 replication

Abstract The replication of herpes simplex virus type 1 (HSV-1) was studied in human embryonic lung cells cultured in the presence of varying concentrations of cytochalasin B. At a concentration of 50 μg/ml, the drug decreased the incorporation of [ 3 H]glucosamine and [ 3 H]fucose into acid-insoluble material by 96 and 92%, respectively, while allowing the incorporation of [ 14 C]amino acids to proceed at a near-normal level. Analysis of [ 14 C]amino acid-labeled HSV-induced polypeptides by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed that as the concentration of cytochalasin B increased, a progressive shift was observed from the major envelope glycopeptide of the virus to a new, presumably nonglycosylated, component designated CB92 (MW 92,000). Concomitant with the appearance of CB92 was a failure of all major virus-induced glycopeptides to incorporate [ 3 H]glucosamine and [ 3 H]fucose. At 50 μg/ml, the drug also decreased HSV-1 infectivity by 96%, although only a 49% reduction of physical particles was observed at this concentration.

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.