Ultrastructural changes during herpes simplex virus type 2 latency and reactivation in vitro.

An in vitro latency system using herpes simplex virus type 2(HSV2)-infected human embryonic lung cells treated with cytosine arabinoside (ara-C) and incubated at 39.5 degrees after drug removal was used to examine the ultrastructure of the infected cells during (i) ara-C treatment, (ii) the latent period after ara-C removal and temperature elevation, and (iii) reactivation after superinfection with human cytomegalovirus. In the presence of ara-C 'empty', nonenveloped, herpesvirus-like particles were observed in nuclei with fragmented chromatin. After removal of ara-C and temperature elevation, no virions were found. At no time during latent infection were complete virions seen. After superinfection with human cytomegalovirus, extensive synthesis of proteins was suggested by a marked proliferation of rough endoplasmic reticulum, polysomes, and Golgi vesicles. This was followed by the appearance of an electron-dense viral matrix in both the nucleus and cytoplasm and by formation of normal nucleocapsids in the nucleus. The nature of the protein(s) required to reactivate HSV2 is unknown but may involve proteins coded by the virus, the host or both.

Herpes simplex virus latency and reactivation in isolated rat sensory neurons.

An in vitro herpes simplex virus type 1 (HSV-1) latency model has been established using neurons isolated from dissociated rat fetus sensory ganglia as the host cell. Rat fetal neuron cells were pretreated for 24 hr at 37 degrees with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon, infected with HSV-1 (approximately 2.5 plaque-forming units/cell), and treated for 7 days with the same inhibitor combination. Infectious HSV-1 became undetectable 3 days postinfection and remained undetectable during the remainder of the inhibitor treatment. After removal of inhibitors on day 7, infectious virus remained undetectable for 2-7 days; subsequently, virus replication ensued and neuronal cells were destroyed. Incubation of inhibitor-treated, infected neuron cells at 40.5 degrees after removal of inhibitors resulted in extension of the latent period to at least 15 days. HSV-1 was reactivated from latently infected neurons by reducing the incubation temperature from 40.5 to 37 degrees and virus-specific cytopathology was observed in neurons within 96 hr after reducing temperature. This in vitro model system will provide the first system to analyze, in a primary cell type of neuronal origin, the state of the HSV genome during establishment and maintenance of the latent state and during virus reactivation.

High efficiency latency and activation of herpes simplex virus in human cells.

Herpes simplex virus (HSV) exists in humans in a latent form that can be activated. To characterize the molecular basis of the cell-virus interactions and to analyze the state of the latent HSV genome, an in vitro model system was established. In this system a large fraction of the latently infected cells contain an HSV genome that can be activated. Cell survival was reduced minimally after repression of high multiplicity HSV type 1 (HSV-1) infection of human fibroblast cells with (E)-5-(2-bromovinyl)-2'-deoxyuridine in combination with human leukocyte interferon (IFN-alpha). A minimum of 1 to 3 percent of the surviving cells contained an HSV genome that could be activated either by human cytomegalovirus superinfection or reduction in incubation temperature.

Activation of herpes simplex virus (HSV) type 1 genome by temperature-sensitive mutants of HSV type 2.

Previous studies have demonstrated that herpes simplex viruses (HSV) type 1 (HSV-1) and type 2 (HSV-2) can be maintained in a repressed form in human embryo lung cells. Reducing the incubation temperature or superinfecting with a heterologous herpesvirus, human cytomegalovirus (HCMV), results in activation of virus replication. We now report that superinfection with a partially homologous herpesvirus, HSV-2, also resulted in activation of HSV-1. To minimize excessive synthesis of infectious HSV-2 while allowing virus gene expression, repressed HSV-l-infected cultures were superinfected with HSV-2 temperature-sensitive (ts) mutants (tsF3, tsB5, or tsH9). The predominant virus replicated after HSV-2 ts mutant superinfection at a nonpermissive temperature was identified as activated parental-like HSV-1 by (i) plaquing efficiency at permissive (34 degrees) and nonpermissive (40.5 degrees) temperatures, (ii) sensitivity to inhibition by the HSV-l-specific antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine, and (iii) restriction endonuclease cleavage analysis. In addition, the fact that superinfection with HSV-2 tsB5 or tsH9, which are unable to synthesize virus DNA and express only early virus genes at nonpermissive temperature, resulted in synthesis of virus demonstrated that HSV-2 DNA synthesis is not required for activation. This system has provided the basis for further studies concerning the regulation of HSV gene expression in human cells.

Repression and activation of the genome of herpes simplex viruses in human cells.

We have described previously a cell culture system in which the herpes simplex virus (HSV) type 2 (HSV-2) genome is maintained in a repressed form after treatment of infected cells with 1-beta-D-arabinofuranosylcytosine and increase of incubation temperature from 37 degrees C to 39.5 degrees C. Infectious HSV-2 production was activated by altering incubation temperature or by superinfecting with human cytomegalovirus. We now report the establishment of an analogous system utilizing HSV type 1 (HSV-1). Human embryo lung cells were infected with HSV-1 and treated with 1-beta-D-arabinofuranosylcytosine (25 micrograms/ml) for 7 days to minimize both synthesis of virus DNA and infectious virus while allowing expression of early virus genes. HSV-1 was maintained in an undetectable form for at least 72 days when the incubation temperature was raised from 37 degrees C to 40.5 degrees C after removal of the inhibitor. HSV-1 gene expression was then predictably turned on by superinfection with human cytomegalovirus or by reducing the incubation temperature. Virus replicated after activation was compared with the respective parental virus with regard to inhibition by the HSV-1-specific antiviral (E)-5-(2-bromovinyl)-2'-deoxyuridine and EcoRI, HindIII, and Xba I restriction endonuclease cleavage patterns. The results show activation of HSV gene expression in human cells by a human cytomegalovirus early gene function(s), followed by synthesis of parental-like HSV.

Inhibition of mouse LM cell replication by trifluorothymidine: role of cytosolic deoxythymidine kinase.

The effects of trifluorothymidine (5-trifluoromethyl-2'-deoxyuridine, F3dThd) on the replication of three mouse cell lines, LM929, Ltk- (and LM929 derivative devoid of cytosolic deoxythymidine [dThd] kinase activity), and Ltk- c139 (a Ltk- derivative which expresses herpes simplex virus type 1-specified dThd kinase subsequent to biochemical transformation with ultraviolet-irradiated herpes simplex virus type 1), have been investigated. Complete inhibition of Ltk- cell growth required a 10,000-fold higher concentration of F3dThd (1.0 mM) than was required to completely inhibit LM929 and Ltk- c139 cell growth. The plating efficiency of exponentially dividing Ltk- cells after exposure to F3dThd (10 microM) for 24 h was 63% as compared to 3% for exponentially dividing LM929 cells. Stationary LM929 cells (confluent cultures held for a 6-day period in serum-reduced medium) with reduced dThd kinase specific activity and deoxyribonucleic acid biosynthesis level exhibited a plating efficiency similar to that of exponentially dividing Ltk- cells after exposure to F3Thd (1.0 mM) for 24 h. In addition, treatment of exponentially dividing LM929 and Ltk- cells with F3dThd (10 microM) for 24 h resulted in approximately an 80% and 25% reduction in deoxyribonucleic acid biosynthesis, respectively. These data indicated a requirement for cytosolic dThd kinase in the expression of F3dThd-induced cytotoxicity. F3dThd was shown to be a linear competitive inhibitor with respect to dThd for affinity-purified LM929 cytosolic dThd kinase. The Km(app) for dThd and Ki(app) for F3dThd with the cytosolic dThd kinase were 2.4 and 3.8 microM, respectively.

HEp-2 cell- and herpes simplex virus type 1- induced deoxythymidine kinases: inhibition by derivatives of 5-trifluoromethyl-2'-deoxyuridine.

5-Trifluoromethyl-2'-deoxyuridine (F(3)Thd), its free base and nucleotide triphosphate derivative, along with the nucleotide monophosphate and nucleotide triphosphate of deoxythymidine (dThd), were investigated as inhibitors of HEp-2 cell deoxythymidine kinase (dTK) and herpes simplex virus type 1 (HSV-1)-induced dTK. 5-Trifluoromethyluracil did not inhibit cellular or viral dTK. F(3)dThd competitively inhibited phosphorylation of dThd by both the HEp-2 cell- and the HSV-1-induced dTK. The K(mapp) for dThd and the K(Iapp) for the alternate substrate, F(3)dThd, were 3.5 and 22.5 muM for the HEp-2 cell dTK and 63.5 and 71.0 muM for the HSV-1-induced dTK. dThd-5'-PPP at 10 muM inhibited HEp-2 cell- and HSV-1-induced dTK by 94 and 22%, respectively. In comparison, 10 muM F(3)dThD-5'-PPP inhibited HEp-2 cell- and HSV-1-induced dTK 95 and 15%, respectively. These data indicate that F(3)dThd-5'-PPP may mimic dThd-5'-PPP feedback regulation of cellular and viral dTK.

Biological effects of 5-carboxy-2'-deoxyuridine: hydrolysis product of 5-trifluoromethyl-2'-deoxyuridine.

5-Carboxy-2'-deoxyuridine (5-COOH-2'-dUrd) is a product of the base-catalyzed hydrolysis of 5-trifluoromethyl-2'-deoxyuridine. Hydrolysis of 5-trifluoromethyl-2'-deoxyuridine to 5-COOH-2'-dUrd in phosphate-buffered saline was kinetically first order and was pH dependent. At 37 degrees C and pH 7.0, 7.5, and 8.0, hydrolysis occurred with rate constants of 4.19 x 10(-5), 9.30 x 10(-5), and 1.61 x 10(-4) s(-1), respectively, with corresponding half-lives of 45.7, 20.6, and 11.9 h. 5-COOH-2'-dUrd inhibited growth of HEp-2 cells by 21, 67, and 91% at 1.0, 10, and 100 muM, with no antiviral activity against herpes simplex virus type 1 or herpes simplex virus type 2 at 1.0 or 10 muM. Partial reversal of cytotoxicity in HEp-2 cells was achieved with orotidine, uridine, deoxythymidine, or deoxycytidine, whereas complete reversal of cytotoxic effects was achieved with simultaneous addition of deoxythymidine, deoxycytidine, and uridine. 5-COOH-2'-dUrd at 50 muM inhibited incorporation of [(14)C]orotate into RNA and DNA by 65 and 27%, respectively. 5-COOH-2'-dUrd had no effect on the incorporation of [(3)H]uridine into DNA or RNA. Because of the structural similarities to deoxythymidine, 5-COOH-2'-dUrd was tested as an inhibitor of deoxythymidine kinase. 5-COOH-2'-dUrd was neither a substrate nor an inhibitor of herpes simplex virus type 1 induced deoxythymidine kinase or HEp-2 cell deoxythymidine kinase. Based on these observations, the metabolic block induced by 5-COOH-2'-dUrd has been localized to the de novo pyrimidine biosynthetic pathway between orotate phosphoribosyl transferase and orotidine 5'-phosphate decarboxylase.