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.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of infected animals. Expression of latency-related (LR) gene products is controlled by a 980-bp fragment (LR promoter).DNA sequence analysis revealed that two major open reading frames (ORFs) are in the LR gene. Antibodies directed against both ORFs were generated in rabbits by using synthetic peptides. Antibody P2, which is directed to sequences near the amino terminus of ORF 2, recognized a 41-kDa protein in lytically infected cells, suggesting that ORF 2 encodes a protein. When the LR gene was inserted into a mammalian expression vector and subsequently transfected into COS-7 cells, a 41-kDa protein was detected by use of silver-stained sodium dodecyl sulfate-polyacrylamide gels and by the P2 antibody. In contrast, this protein was not detected in mock-transfected cells. Deletion of DNA sequences containing ORF 2 blocked synthesis of the 41-kDa protein in COS-7 cells. Reverse transcriptase-mediated PCRs indicated that splicing occurs near the C terminus of ORF 2. Further studies indicated that LR RNA was alternatively spliced in latently infected cattle and that a fraction of LR RNA was poly(A) ؉ . Taken together, these studies suggested that a spliced LR transcript has the potential to encode a 41-kDa protein.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in the sensory ganglionic neurons of cattle. The exclusive viral RNA expressed in a latent infection is the latency-related (LR) RNA, suggesting that it regulates some aspect of a latent infection. During the course of a productive infection, alphaherpesviruses induce certain events which occur during cell cycle progression. Consequently, we hypothesized that a BHV-1 infection might induce events in neurons which occur during cell cycle progression. In agreement with this hypothesis, cyclin A was detected in neurons of trigeminal ganglia when rabbits were infected. Neuronal cell cycle progression or inappropriate expression of cyclin A leads to apoptosis, suggesting that a viral factor inhibits the deleterious effects of cyclin A expression. The BHV-1 LR gene inhibited cell cycle progression and proliferation of human osteosarcoma cells. Antibodies directed against cyclin A or the LR protein coprecipitated the LR protein or cyclin A, respectively, suggesting that the two proteins interact with each other. We conclude that LR gene products inhibit cell cycle progression and hypothesize that this activity enhances the survival of infected neurons.

During an infection of nonneuronal cells, bovine herpesvirus 1 (BHV-1) gene expression proceeds in a well-defined cascade. Products of immediate-early (IE) genes are expressed first, and they activate expression of early (E) and late (L) genes. Although the same cascade is assumed to occur during an infection of neurons in trigeminal ganglia (TG) of cattle, no experimental data is available to support this hypothesis. Consequently, we analyzed BHV-1 gene expression in bovine TG at 1, 2, 4, 7, and 15 days postinfection (dpi). Infectious virus was detected in ocular swabs from 1 to 7 dpi but not 15 dpi. By reverse transcription (RT)-PCR, IE (bICP4), E (thymidine kinase, ribonucleotide reductase [RR]), L (glycoprotein C, and ␣ trans-inducing factor), and dual-kinetic (bICP0 and bICP22) transcripts were analyzed. When cDNA synthesis was primed with random hexamers, IE and E transcripts were detected at the same time. However, full-length and poly(A) ؉ (FL&P) RR or bICP22 RNAs were detected before FL&P IE RNAs. Furthermore, FL&P IE transcripts were not detected until viral DNA increased in TG. IE transcripts were detected before E or L RNAs when rabbit kidney cells were infected with a low multiplicity of infection and the same RT-PCR detection method was used. These studies suggested that expression of full-length and polyadenylated IE transcripts in trigeminal ganglia was not efficient compared to that of RR and bICP22 transcripts.

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.