Troy Lund

In vitro antiviral drug sensitivity of the Kaposi's sarcoma-associated herpesvirus

Objective:Kaposis sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, has been implicated as the causative agent of Kaposis sarcoma. Retrospective studies show that the risk of development of Kaposis sarcoma is significantly lower in AIDS patients who received ganciclovir or phosphonoformic acid (PFA) therapy. Therefore, in vitro antiviral drug sensitivity of KSHV was studied. Methods:The KSHV genome is a latent episome in lymphoma cells such as the BCBL-1 cell line. Lytic KSHV DNA synthesis is induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in BCBL-1 cells; this system was used to evaluate the effects of antiviral drugs on KSHV DNA synthesis. Results:Linear (lytic) KSHV DNA synthesis and virus secretion was inhibited in BCBL-1 cell cultures by cidofovir (median inhibitory concentration, 0.05 µM), ganciclovir (5.1 µM) and PFA (97 µM), and by aciclovir (75 µM). Prolonged incubation of BCBL-1 cells with antiviral drugs had no effect on episomal KSHV DNA synthesis. Conclusions:The antiviral drug assay developed shows that KSHV is very sensitive to cidofovir, moderately sensitive to ganciclovir and PFA, and weakly sensitive to aciclovir. Therefore, low doses of cidofovir, or high doses of PFA or ganciclovir could suppress clinical reactivation of KSHV. Antiviral drugs did not inhibit episomal virus DNA synthesis, suggesting that the latent form of viral DNA is replicated by host DNA polymerases. Consequently, no benefit can be expected from antiviral drugs in KSHV-positive B-cell lymphomas or during latency.

Activation of the Lck Tyrosine-protein Kinase by the Binding of the Tip Protein of Herpesvirus Saimiri in the Absence of Regulatory Tyrosine Phosphorylation

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip eitherin vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.

The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome

The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposis sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.

The Src-family kinase Lck can induce STAT3 phosphorylation and DNA binding activity.

Constitutive activation of the Src-family kinase Lck has been shown to lead to transformation. Constitutive activation of the STAT pathway of transcription factors has also been shown to be involved in transformation. An oncogenic form of the prototypical member of the Src-family, v-Src, has been shown to activate STAT3, and this activation is required for v-Srcs transforming ability. To investigate whether Lck could directly activate STAT3, a baculovirus expression system was utilised. When Lck and STAT3 were coexpressed, STAT3 was found to have enhanced tyrosine phosphorylation and DNA binding activity. This finding was confirmed with experiments where exogenous Lck was added to baculovirus produced STAT3. Moreover, the activation of STAT3 by exogenous Lck could be attenuated by the Lck-specific inhibitor PP1. In addition, mammalian cells stably expressing a constitutively activated form of Lck were shown to have activated STAT3. These data provide strong evidence that, like v-Src, Lck can also directly activate STAT3, which contributes to the transformation process.

A polycistronic transcript in transformed cells encodes the dihydrofolate reductase of herpesvirus saimiri

Herpesvirus saimiri, an oncogenic gamma herpesvirus of primates, is the only eukaryotic virus that carries the entire metabolic gene set for a complex biochemical synthesis. Every element of the thymidine synthesis gene cascade is present in the virus, and their function is probably related to the uniquely high A + T content of the genome. Although one member of the gene set, dihydrofolate reductase (DHFR), is mapped in a region required for oncogenesis, very little is known of the expression and function of this gene in transformed cells. We report the expression of the DHFR sequence on a novel, unique tricistronic transcript in virally transformed tumor cells. The DHFR sequence is the first open reading frame on a 5.3 kb minor transcript. Alpha-amanitine sensitivity indicates that it is an RNA polymerase II transcript, and since it is also polyadenylated it appears to be a functional, relatively unstable (half-life 3 hr) mRNA. Initiation of transcription uniquely overlaps with the HSUR3 small RNA gene. Expression of the small transcript appears to be alpha-amanitine resistant, implicating polymerase III transcription. Together with the remarkably low-level expression of HSUR3 in tumor cells, the data may indicate transcription interference between two different RNA polymerases, with unusual overlapping regulation and initiation.