Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants

Self Cite

The buoyant density characteristics of infectious particles of herpes simplex virus types 1 and 2 were studied by centrifugation in sucrose and cesium chloride density gradients with a high resolution and satisfactory infectivity recovery. It was shown that two populations of infectious virions differing in buoyant density coexisted, the difference being slight but definite. The ratio of heavy (H) to light (L) viral particles varied depending upon the solute used, the strain of virus, and the cell origin. Circumstances favoring degradation of viral infectivity tended to increase the H portion. Incubation at 37°C largely converted L to H, and heating at 450C converted all virions to H without infectivity. The L to H conversion was irreversible, and no populations intermediate between L and H were clearly observed. Inactivation by UV light irradiation did not affect the density pattern.

Section: Methodsmentioning

confidence: 99%

Irreversible conversion of the physical state of herpes simplex virus preceding inactivation by thermal or antibody treatment

Yanagi¹

1981

Self Cite

“…Indeed, relationships between HSV infection and cell cyclerelated cellular functions are well documented. Thus, HSV replication is blocked at the nonpermissive temperature in five temperature-sensitive cell lines growth arrested in G 0 /G 1 (55,61). Moreover, HSV has long been known to replicate more efficiently in actively dividing than in growth-arrested cells of most types, and this enhancement of replication efficiency is especially prominent for certain HSV strains with mutations in genes not absolutely required for viral replication (5,10).…”

mentioning

confidence: 99%

Requirement for Cellular Cyclin-Dependent Kinases in Herpes Simplex Virus Replication and Transcription

Schang

Schaffer

1998

145

Several observations indicate that late-G1/S-phase-specific cellular functions may be required for herpes simplex virus (HSV) replication: (i) certain mutant HSV strains are replication impaired during infection of cells in the G0/G1 but not in the G1/S phase of the cell cycle, (ii) several late-G1/S-phase-specific cellular proteins and functions are induced during infection, and (iii) the activity of a cellular protein essential for expression of viral immediate-early (IE) genes, HCF, is normally required during the late G1/S phase of the cell cycle. To test the hypothesis that late-G1/S-phase-specific cellular functions are necessary for HSV replication, HEL or Vero cells were infected in the presence of the cell cycle inhibitors roscovitine (Rosco) and olomoucine (Olo). Both drugs inhibit cyclin-dependent kinase 1 (cdk-1) and cdk-2 (required for cell cycle progression into the late G1/S phase) and cdk-5 (inactive in cycling cells) but not cdk-4 or cdk-6 (active at early G1). We found that HSV replication was inhibited by Rosco and Olo but not by lovastatin (a cell cycle inhibitor that does not inhibit cdk activity), staurosporine (a broad-spectrum protein serine-threonine kinase inhibitor), PD98059 (an inhibitor specific for erk-1 and -2) or iso-Olo (a structural isomer of Olo that does not inhibit cdk activity). The concentrations of Rosco and Olo required to inhibit cell cycle progression and viral replication in both HEL and Vero cells were similar. Inhibition of viral replication was found not to be mediated by drug-induced cytotoxicity. Efforts to isolate Rosco- or Olo-resistant HSV mutants were unsuccessful, indicating that these drugs do not act by inhibiting a single viral target. Viral DNA replication and accumulation of IE and early viral RNAs were inhibited in the presence of cell cycle-inhibitory concentrations of Rosco or Olo. We therefore conclude that one or more cdks active from late G1 onward or inactive in nonneuronal cells are required for accumulation of HSV transcripts, viral DNA replication, and production of infectious virus.

“…The cellular protein defective in one of these ts cell lines has been identified as HCF, which is required for binding of a viral transactivator, VP16, to viral immediate-early (IE) promoters. Thus, in addition to its previously recognized role in HSV replication (16,67), HCF is an important regulator of cell cycle progression (16,65). The cellular proteins defective in other ts cell lines that arrest in G 0 /G 1 and do not support HSV replication have not yet been characterized but may potentially include any of the cellular proteins involved in cell cycle progression that are also (i) required for efficient HSV replication (16,20), (ii) activated during HSV infection (25,30), (iii) localized to the sites of viral replication (11,64), and/or (iv) interactive physically with HSV DNA replication proteins (34).…”

mentioning

confidence: 95%

“…The phenotypes of these mutants suggest that one of the functions of these viral proteins is to induce or replace cellular activities which are normally activated in a cell-cycle-regulated manner. In addition to the impaired replication efficiency of ICP0 and VP16 mutants in noncycling cells, wild-type HSV cannot replicate at the nonpermissive temperature in several temperature-sensitive (ts) cell lines that are growth arrested in G 0 /G 1 , implying that viral replication requires one or more cellular functions activated in a cellcycle-dependent manner in noninfected cells (62,67). The cellular protein defective in one of these ts cell lines has been identified as HCF, which is required for binding of a viral transactivator, VP16, to viral immediate-early (IE) promoters.…”

mentioning

confidence: 99%

Transcription of Herpes Simplex Virus Immediate-Early and Early Genes Is Inhibited by Roscovitine, an Inhibitor Specific for Cellular Cyclin-Dependent Kinases

Schang

Rosenberg

Schaffer

1999

Although herpes simplex virus (HSV) replicates in noncycling as well as cycling cells, including terminally differentiated neurons, it has recently been shown that viral replication requires the activities of cellular cyclin-dependent kinases (cdks) (L. M. Schang, J. Phillips, and P. A. Schaffer, J. Virol. 72:5626–5637, 1998). Since we were unable to isolate HSV mutants resistant to two cdk inhibitors, Olomoucine and Roscovitine (Rosco), we hypothesized that cdks may be required for more than one viral function during HSV replication. In the experiments presented here, we tested this hypothesis by measuring the efficiency of (i) viral replication; (ii) expression of selected immediate-early (IE) (ICP0 and ICP4), early (E) (ICP8 and TK), and late (L) (gC) genes; and (iii) viral DNA synthesis in infected cultures to which Rosco was added after IE or IE and E proteins had already been synthesized. Rosco inhibited HSV replication, transcription of IE and E genes, and viral DNA synthesis when added at 1, 2, or 6 h postinfection or after release from a 6-h cycloheximide block. Transcription of a representative L gene, gC, was also inhibited by Rosco under all conditions examined. We conclude from these studies that cellular cdks are required for transcription of E as well as IE genes. In contrast, steady-state levels of at least one cellular housekeeping gene were not affected by Rosco. The requirement of viral IE and E transcription for cellular cdks may reflect either a requirement for specific cdk-activated cellular and/or viral transcription factors or a more global requirement for cdks in the transcriptional activation of the viral genome.