Biomedical subjects

L M Schang

Publications and source records attributed to L M Schang.

12 recordsLinked to original sources

PubMed2001

Cellular proteins (cyclin dependent kinases) as potential targets for antiviral drugs.

Several pharmacological inhibitors that are specific for cellular cyclin-dependent kinases (cdks) have recently been found to repress viral replication in vitro. Thus, replication of human cytomegalovirus, herpes simplex virus, and HIV-1 is repressed by pharmacological cdk inhibitors (PCIs). Replication of several other viruses requires cdks that are sensitive to PCIs. Interestingly, some of the PCIs that have antiviral activity in vitro are being tested in animal models and in human clinical trials as anti-cancer agents, showing little toxicity in vivo. Thus, PCIs have the potential to become antiviral drugs for clinical use. As PCIs repress viral replication by targetting cellular proteins, they would constitute a novel group of antivirals. They could be active against several unrelated viruses, and viral mutants that are resistant to conventional antiviral drugs, and could be used in combination with any antiviral drug that targets a viral protein. For viral diseases whose pathological mechanism requires cdks, such as virus-induced tumours, PCIs would repress both the aetiological agent and the pathogenic mechanism. In this review, the biochemical, cellular and antiviral activities of PCIs in vitro and their toxicity in vivo are discussed. Other cellular proteins that are required for viral replication could also be targets for new antiviral drugs.

Animals↗

PubMed2000

Human PC4 is a substrate-specific inhibitor of RNA polymerase II phosphorylation.

The activity of cyclin-dependent protein kinases (cdks) is physiologically regulated by phosphorylation, association with the specific cyclin subunits, and repression by specific cdk inhibitors. All three physiological regulatory mechanisms are specific for one or more cdks, but none is known to be substrate specific. In contrast, synthetic cdk peptide inhibitors that specifically inhibit cdk phosphorylation of only some substrates, "aptamers," have been described. Here, we show that PC4, a naturally occurring transcriptional coactivator, competitively inhibits cdk-1, -2, and -7-mediated phosphorylation of the largest subunit of RNA polymerase II (RNAPII), but it does not inhibit phosphorylation of other substrates of the same kinases. Interestingly, the phosphorylated form of PC4 is devoid of kinase inhibitory activity. We also show that wild-type PC4 but not the kinase inhibitory-deficient mutant of PC4 represses transcription in vivo. Our results point to a novel role for PC4 as a specific inhibitor of RNAPII phosphorylation.

Cell Line↗

PubMed2000

Roscovitine, a specific inhibitor of cellular cyclin-dependent kinases, inhibits herpes simplex virus DNA synthesis in the presence of viral early proteins.

We have previously shown that two inhibitors specific for cellular cyclin-dependent kinases (cdks), Roscovitine (Rosco) and Olomoucine (Olo), block the replication of herpes simplex virus (HSV). Based on these results, we demonstrated that HSV replication requires cellular cdks that are sensitive to these drugs (L. M. Schang, J. Phillips, and P. A. Schaffer. J. Virol. 72:5626-5637, 1998). We further established that at least two distinct steps in the viral replication cycle require cdks: transcription of immediate-early (IE) genes and transcription of early (E) genes (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Since Rosco inhibits HSV replication efficiently even when added to infected cells at 6 h postinfection, we postulated that cdks may also be required for viral functions that occur after E gene expression. In the study presented herein, we tested this hypothesis directly by measuring the efficiency of viral replication, viral DNA synthesis, and expression of several viral genes during infections in which Rosco was added after E proteins had already been synthesized. Rosco inhibited HSV replication, and specifically viral DNA synthesis, when the drug was added at the time of release from a 12-h phosphonoacetic acid (PAA)-induced block in viral DNA synthesis. Inhibition of DNA synthesis was not a consequence of inhibition of expression of IE or E genes in that Rosco had no effect on steady-state levels of two E transcripts under the same conditions in which it inhibited viral DNA synthesis. Moreover, viral DNA synthesis was inhibited by Rosco even in the absence of protein synthesis. In a second series of experiments, the replication of four HSV mutants harboring temperature-sensitive mutations in genes essential for viral DNA replication was inhibited when Rosco was added at the time of shift-down from the nonpermissive to the permissive temperature. Viral DNA synthesis was inhibited by Rosco under these conditions, whereas expression of viral E genes was not affected. We conclude that cellular Rosco-sensitive cdks are required for replication of viral DNA in the presence of viral E proteins. This requirement may indicate that HSV DNA synthesis is functionally linked to transcription, which requires cdks, or that both viral transcription and DNA replication, independently, require viral or cellular factors activated by Rosco-sensitive cdks.

Animals↗

PubMed1999

Transcription of herpes simplex virus immediate-early and early genes is inhibited by roscovitine, an inhibitor specific for cellular cyclin-dependent kinases.

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.

Animals↗

PubMed1998

Requirement for cellular cyclin-dependent kinases in herpes simplex virus replication and transcription.

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.

Amino Acid Sequence↗

PubMed1997

Analysis of bovine herpesvirus 1 transcripts during a primary infection of trigeminal ganglia of cattle.

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 alpha 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.

Acute Disease↗

PubMed1996

The latency-related gene of bovine herpesvirus 1 encodes a product which inhibits cell cycle progression.

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.

Animals↗

PubMed1995

Identification of gene products encoded by the latency-related gene of bovine herpesvirus 1.

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.

Amino Acid Sequence↗

PubMed1994

Quantitation of latency established by attenuated strains of Pseudorabies (Aujeszky's disease) virus.

A quantitative and differential polymerase chain reaction (PCR) was developed that measures the ability of Pseudorabies virus (PRV) to colonize tissues that are targets for latency. This PCR is based on the co-amplification of viral target sequences and that of a gene of the host species: the porcine Nuclear Factor 1 gene, which serves as an internal standard. The technique was found to be sensitive and reproducible. Using this technique, individual variabilities were detected in the level of colonization of trigeminal ganglia established by a PRV strain among different animals. At a population level, it was established that two different PRV attenuated strains establish latency with different efficiencies, when applied by the same route and at similar inoculation dose.

Animals↗

PubMed1994

Correlation between precolonization of trigeminal ganglia by attenuated strains of pseudorabies virus and resistance to wild-type virus latency.

We compared the levels of latent pseudorabies virus (PRV) DNA in trigeminal ganglia (TG) of pigs after intranasal inoculation of different PRV strains by using quantitative DNA PCR. The extent of colonization attained in each case varied significantly according to the type of strain and inoculum dose, wild-type (WT) PRV being the most efficient strain in colonizing TG. When groups of pigs representing different levels of precolonization of TG with an attenuated PRV strain were challenged with WT PRV, it became evident that there is a statistically significant inverse correlation between the extent of precolonization attained by an attenuated PRV strain in TG and the level of establishment of latency by superinfecting WT PRV. The protection against WT PRV latency did not correlate with the extent of WT PRV replication at the portal of entry.

Analysis of Variance↗

PubMed1993

A quantitative technique for the study of the latency of Aujeszky virus.

Latency represents a challenge for any herpesvirus vaccine. Vaccines could differ in their ability to minimize latency of pseudorabies virus (PRV). To study this possibility, a quantitative and differential PRV-specific polymerase chain reaction (PCR) was developed by the authors. Efficiency of amplification in different tubes is measured by co-amplification with the viral targets (either gI or gp50 genes) of the porcine gene nuclear factor 1. The criteria used to select this approach to a quantitative PCR, as well as for the selection of the standard, are discussed. The amplified products are measured by Southern blot or, alternatively, high performance liquid chromatography. Sensitivity and reproducibility of the technique are evaluated. The ability of this technique to discriminate between the level of latency established by two different PRV strains in trigeminal ganglia is also evaluated. Using this technique, the authors are currently studying whether different vaccines could possess differing levels of ability to minimize, by interference, establishment of wild-type latency.

Animals↗

PubMed1991

Influence of antioxidants on SOD activity in bovine sperm.

SOD activity and susceptibility to peroxidation on spermatozoa from frozen and fresh bovine semen were determined either in presence or not of synthetic (BHA) or natural (vitamin E) antioxidants. In sperm suspensions incubated with vitamin E, SOD activity was higher than in the control samples and the ones treated with BHA. It was found a highly significative correlation between malondialdehyde production and SOD activity. SOD activity could be used in bovine spermatozoa as a metabolic indicator of membrane integrity.

Animals↗