Yung-Chi Cheng

Mode of action of phosphonoformate as an anti-herpes simplex virus agent

Phosphonoformate inhibited the replication of Herpes simplex virus (HSV) type 1 and type 2 in culture. The concentration required to inhibit the replication of both types of virus by 2 logs at 28 h post-infection was approximately 150 microM. It was more potent than phosphonoacetate against the growth of both virus types. A virus mutant which is resistant to phosphonoacetate was cross-resistant to phosphonoformate. Arsonoacetate, at 300 microM, had no antivirus activity. Phosphonoformate also inhibited HeLa and KB cell growth; at a concentration of about 500 microM, cell growth was inhibited by 50%. The anti-cell growth effects of the drug were completely reversible. The antivirus effect of phosphonoformate was partially reversible, depending on the time and duration of exposure of infected cultures to the drug. To obtain the maximum antivirus effect, phosphonoformate had to be added within the first 3 h post-virus-infection and be continuously present for at least 18 h. Phosphonoformate, added at 0 h post-infection, suppressed the induction of virus-specific DNA polymerase and DNAase activities. dTMP incorporation into DNA was preferentially inhibited in nuclei isolated from infected cells compared to uninfected cells, and the degree of inhibition varied with the ionic strength of the assay. Phosphonoformate was a potent inhibitor of the purified HSV-1 and HSV-2 DNA polymerases, inhibiting DNA polymerase activity by 50% at a concentration of 3 microM and ionic strength of 0.2.

The intracellular content of dihydrofolate reductase: possibilities for control and implications for chemotherapy.

Intracellular levels of DHFR can be modulated by mechanisms other than gene amplification. We found that MTX itself has an effect and the important features of this mechanism are as follows: (a) Sub-saturating doses of MTX induce intracellular DHFR activity by increasing DHFR synthesis; (b) The time-dependent effect seems quite specific for DHFR and is reversible (7); (c) Elevated DHFR synthesis is accompanied by disproportionate increases in DHFR mRNA; (d) The time scale for maximum induction is appreciably longer than the cell generation time. We suggest that part of the control involved is translational and we postulate that DHFR may regulate its own biosynthesis through feedback mechanisms. It is conceivable that the induction phenomenon could affect the clinical efficacy of MTX-therapy in some instances.

Can ribonucleotide reductase be considered as an effective target for developing antiherpes simplex virus type II (HSV-2) compounds?

Herpes simplex viruses are known to induce virus specified ribonucleotide reductase (RR) in infected cells. RR is considered as a possible target for the development of antiviral agents. In this study, the role of RR in virus replication has been investigated. The sensitivity of RR to hydroxyurea (HU) from virus infected and uninfected HeLa S3 cells was similar with IC50 values of 0.12 and 0.14 mM. In the presence of 2 mM HU, and 10 microM tetrahydrouridine (THU), a cytidine deaminase inhibitor, the incorporation of [14C]cytidine into viral DNA was found to be inhibited by 95%; [32P]-incorporation into viral DNA under the same conditions was inhibited by 75%. The pool size of dCTP and dGTP was 50 and 70%, respectively, with no significant effect on dATP and dTTP pools in virus infected cells treated with 2 mM HU, as compared with virus infected cells receiving no drug treatment. HU at 2 mM could not inhibit HSV-2 yield by more than one log. These results suggest that virus RR is not an effective target for developing anti HSV-2 compounds.

Characterization of a novel inhibitor of human DNA polymerases: 3,4,5-tri-O-galloylquinic acid☆

Various galloyl derivatives of quinic acid were found to be inhibitors of human DNA polymerases. Among them, 3,4,5-tri-O-galloylquinic acid (TGQA) was the most potent inhibitor of DNA polymerase alpha. Under identical conditions, this compound was 60-fold more potent than aphidicolin as an inhibitor of DNA polymerase alpha. The inhibition of DNA polymerase alpha by this compound was not competitive with either the template or any of the deoxynucleoside triphosphates with a Ki of 0.28 microM. Under similar reaction conditions, DNA polymerases beta and gamma were much less sensitive to the effects of these compounds and, in contrast to the effect seen with DNA polymerase alpha, the inhibition of DNA polymerases beta and gamma by TGQA was competitive with respect to the template with Ki values of 44.4 and 7.5 microM respectively. The potency of these compounds against DNA polymerase gamma varied according to the assay conditions used. The inhibition of DNA polymerase gamma by TGQA could be increased substantially by using MnCl2 in place of MgCl2 and by including 50 mM potassium phosphate, pH 7.5, in the assay mixture. DNA polymerase beta was also more sensitive to TGQA when measured with MnCl2. However, potassium phosphate had little, if any, effect on the inhibition by TGQA of either DNA polymerase alpha or beta. DNA polymerase alpha was less sensitive to TGQA when assayed with MnCl2. TGQA was not a potent inhibitor of human KB cell growth in culture, which could be due to its degradation or poor uptake. Nevertheless, this compound could serve as a model for developing antitumor drugs targeted at DNA polymerases.

Studies on DNA Topoisomerases I and II in Herpes Simplex Virus Type 2-infected Cells

It has been suggested that herpes simplex virus (HSV) type 1 may induce a virus-specific DNA topoisomerase activity which copurifies with virus-induced DNA polymerase. We have examined DNA topoisomerase (TOPO) I and II activities in HSV-2-infected HeLa S3 cells. Both activities were partially purified using DEAE-cellulose, phosphocellulose and double-stranded DNA cellulose column chromatography. It was found that both activities could be separated from HSV-2-specific DNA polymerase. Throughout the purification TOPO I could be immunologically detected with a monoclonal antibody developed against human TOPO I. Regardless of the source, mock- or HSV-2-infected human cells, both types of topoisomerase were equally tolerant of 200 mM-KCl. There appeared to be no apparent heterogeneity of TOPO I in HeLa S3 cells through the course of the HSV-2 infection. We conclude that host cell topoisomerases are quite stable in HSV-2-infected HeLa S3 cells and that there is no evidence that HSV-2 is capable of inducing HSV-2-specific TOPO I and TOPO II activities.

Synthesis of dihydrofolate reductase and metabolism of related RNA in a methotrexate resistant human cell line infected with herpes simplex virus type 2.

Using a human cell line with amplified gene copy for dihydrofolate reductase (DHFR) and permissive for herpes simplex virus type 2 (HSV-2), the effect of HSV-2 on DHFR synthesis and on the steady-state level of total cellular and nuclear DHFR RNA was examined. There was a reduction in DHFR synthesis accompanied by a proportional decrease in the levels of DHFR messenger RNA (mRNA) 1 hr after infection. Both effects could be induced by HSV pretreated with ultraviolet light and this early virus induced rapid turnover of DHFR mRNA was not dependent on de novo protein synthesis. Analysis of nuclear RNA (nRNA) from uninfected cells by Northern blot hybridization identified a large DHFR nRNA of about 23 kb which probably represents the primary transcript and four processed intermediates of DHFR mRNA ranging in size from 12 to 4.4 kb. The levels of these nRNAs were unchanged during the first hour; however, the 4.4-kb species accumulated in nuclei 2 hr after infection. This effect was induced in the absence of HSV gene expression.

Study of ribonucleotide reductase in cells infected with six clinical isolates of herpes simplex virus type 2 (HSV-2) with mutations in its larger subunit

Herpes simplex virus type 2 (HSV-2) induces a novel ribonucleotide reductase (RR) composed of two subunits (140 and 38 kDa) in infected cells. Other investigators have developed a monoclonal antibody, A6, against the 140-kDa subunit of RR and have found, in about 1% of the cases, an inability to detect this protein in cells infected with clinical isolates of HSV-2. We therefore investigated whether in such cases the clinical isolates were capable of inducing viral RR activity and whether the lack of detection of the 140-kDa protein by the monoclonal antibody was due to an alteration in the antigenic site of this protein. Six such isolates were examined and were found to induce RR activity, similar to HSV-2 (strain 333) RR, which did not require ATP for CDP reduction. Western blot analyses using A6 failed to detect the protein. However, R1, a polyclonal antibody raised against viral RR was capable of detecting this subunit. In addition, R1 was also capable of neutralizing RR activity induced by all the isolates and HSV-2 (strain 333). In conclusion, the lack of detection of the large subunit of RR was not due to the lack of induction but was due to an alteration in the antigenic site recognized by A6; this alteration did not appear to affect the properties of the induced RR activity.

Development of Antiherpes Virus Agents on the Basis of Virus Induced Enzymes

Herpes Viruses induce many disease entities in humans. Unfortunately, the drugs currently used in the clinical treatment of herpes virus infectious diseases all have substantial toxicity (1,2,3). Recently, several nucleoside analogs such as AraA, 5-alkyl dUrd and Acycloguanine were shown to be effective against herpes simplex virus type 1 infectious diseases without having much toxicity in humans (4–10) however, they all have their own limitation of use. For example, 5-alkyl-dUrd and acycloguanosine will not be active against herpes virus which cannot induce thier virus specific thymidine kinase (TK) (6,11) or herpes virus which could induce a virus specific TK but cannot recognize either compound as its substrate. AraA toxicity will be affected by the amount of adenosine deaminase in human cells. Therefore, it is important to develop more specific antiherpes virus agents for the treatment of herpes virus induced diseases, particularly in patients undergoing cancer chemotherapy or with immune deficiencies.

Enhancement of herpes simplex virus type 2 (HSV-2) DNA synthesis in infected cells that constitutively express the BglII-N region of the HSV-2 genome

The BglII-N fragment of the herpes simplex virus type-2 (HSV-2) genome encodes one of two known transforming regions of this DNA virus. In this study, we report the derivation of HeLa S3 cells (2DC4) that stably express the HSV-2 BglII-N region, including the small subunit of HSV-2 ribonucleotide reductase (RR). Superinfection of the 2DC4 cells with wild-type HSV-2 resulted in the efficient induction of HSV-2-encoded ICP10, DNA polymerase, and thymidine kinase. The amount of HSV-2 DNA synthesis in 8-hr HSV-2-infected 2DC4 cells was enhanced 2.6±0.6-fold relative to infected control cells. Furthermore, the replication kinetics of HSV-2 DNA in 2DC4 cells were accelerated relative to HeLa S3 cells; HSV-2 DNA synthesis was detectable as early as 3 hr postinfection in 2DC4 cells as compared to 6 hr postinfection in HeLa S3 cells. These results suggest that the BglII-N region of HSV-2 encodes function(s) that activate the viral DNA synthesis apparatus and that this activation could relate to the transforming ability of this DNA region.

Human topoisomerase 1 messenger RNA is not destabilized by the herpes simplex virus type 2 virion-associated shut-off function

A cDNA for human topoisomerase I (Topo 1) was used to identify a 4.1 kb polyadenylated Topo 1 mRNA in methotrexate-resistant human KB cells that are permissive for herpes simplex virus type 2 (HSV-2) infection. Using these cells, no effect of the HSV-2-associated early shut-off function on levels of Topo-1 mRNA was observed up to 6 hours postinfection, whereas the actin mRNA level was 22% cellular transcripts are susceptible. The level of several host-cell polyadenylated RNAs detected as cDNA clones (class 3 transcripts) were unchanged 8 hours after HSV-2 infection, and other cellular transcripts (class 2) actually accumulated at postinfection.