A guanosine analog, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG), was found to inhibit herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, cytomegalovirus, and Epstein-Barr virus replication by >50% at concentrations that do not inhibit cell growth in culture. The potency of the drug against all of these viruses is greater than that of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir). DHPG was active against HSV-1 growth during the early phase of virus replication and had no activity when added at a later time after infection. Its antiviral activity was irreversible. Thymidine partially neutralized its action. The anti-HSV-1 activity of DHPG was dependent on the induction and the properties -of virus-induced thymidine kinase. Virus variants that induced altered virus thymidine kinase and became resistant to acyclovir were still as sensitive to DHPG as the parental virus. DHPG is active against five different HSV variants with induced altered DNA polymerase and resistance to acyclovir. Cells infected by herpesviruses, which are important causes of human diseases, undergo many biochemical changes, among which are changes in several virus-specified enzymes that are involved in either deoxynucleotide or DNA metabolism (1). Nucleoside analogs with unique behavior toward such enzymes have been discovered to have selective anti-herpesvirus activity (2-9). Among the nucleoside analogs discovered, 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), one of the new nucleoside analogs with such activity, was recently licensed in the United States for the treatment of primary genital herpesvirus infection and disseminated herpetic infection. Some of the other compounds are at different stages of clinical development. Recently, an analog of guanosine, 9-[(1,3-dihydroxy-2-propoxy)-methyl]guanine (DHPG), with the structure shown in Fig. 1, was synthesized independently by Syntex (Palo Alto, CA) (10) and Biologics (Toronto, ON, Canada) (11) and was found to have anti-herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) activity. HSV that lacked the ability to induce virally coded thymidine kinase (TK) was found to be resistant to DHPG. Nevertheless, phosphonoacetate-resistant HSV, which is crossresistant to acyclovir, was as sensitive to DHPG as the parental virus, whereas a marginal activity of DHPG against Epstein-Barr virus (EBV) and no activity against human cytomegalovirus (CMV) were reported (11).In view of the potency and the unique spectrum of antiviral activity of DHPG, we examined: (i) the activity of this compound against several unique HSV mutants that had either altered TK or DNA polymerase; (ii) its behavior toward HSV-induced TK and its mode of action against HSV-1; and (iii) sensitivity of several strains of CMV and EBV to the drug. The results of the studies are reported in this communication.

The Epstein-Barr virus early antigen diffuse component (EA-D) is essential for Epstein-Barr virus DNA polymerase activity, and its activity is suppressed during latent infection. We investigated the regulation of the promoter (BMRFl) for this early gene by studying its responsiveness in vitro to two immediate-early viral transactivators, BZLF1 (Z) and BRLF1 (R), focusing on the differences in response in lymphoid cells and epithelial cells. In lymphoid cells, Z or R alone produced only small increases in EA-D promoter activity, whereas both transactivators together produced a large stimulatory effect. In epithelial cells, the Z transactivator alone produced maximal stimulation of the EA-D promoter; the effect of R and Z together was no greater than that of Z alone. Deletional analysis and site-directed mutagenesis of the EA-D promoter demonstrated that in epithelial cells the potential AP-1 binding site plays an essential role in Z responsiveness, although sequences further upstream are also important. In lymphoid cells, only the upstream sequences are required for transactivation by the Z/R combination, and the AP-1 site is dispensable. These data suggest that EA-D (BMRF1) promoter regulation by Z and R is cell type specific and appears to involve different mechanisms in each cell type. MATERIALS AND METHODS Cell lines. The cell lines used include the EBV-negative Burkitt's lymphoma B-cell line (Louckes), a T-cell line 3753

We have previously shown that the Epstein-Barr virus (EBV) immediate-early gene product, BZLF1, can activate expression of the EBV BMLF1 immediate-early promoter in EBV-positive, but not EBV-negative, B cells, suggesting that the BZLF1 effect may be mediated through another EBV gene product (S. Kenney, J. Kamine, E. Holley-Guthrie, J.-C. Lin, E.-C. Mar, and J. S. Pagano, J. Virol. 63:1729-1736, 1989). Here, we show that the EBV BRLF1 immediate-early gene product transactivates the BMLF1 promoter in either EBV-positive or EBV-negative B cells. Deletional analysis revealed that both the BZLF1-responsive region and the BRLF1-responsive region of the BMLF1 promoter are contained within the same 140-base-pair FokI-PvuII fragment located 300 base pairs upstream of the mRNA start site. This FokI-PvuII fragment functions as an enhancer element in the presence of the BRLF1 transactivator and contains the sequence CCGTGGAGA ATGTC, which is strikingly similar to the BRLF1-responsive region of the EBV DR/DL enhancer (A. Chevallier-Greco, H. Gruffat, E. Manet, A. Calender, and A. Sergeant, J. Virol. 63:615-623, 1989). The effect of BZLF1 on the BMLF1 promoter is likely to be indirect and mediated through the BRLF1 transactivator.

Glycoproteins M (gM) and N (gN) are well conserved across the herpesvirus family and their involvement in virus penetration and egress is well described, especially for alphaherpesviruses. Because there was no previous study on the homologues of human herpesvirus 8 glycoproteins M (gM8) and N (gN8), we analysed their biochemical and functional characteristics. We found that: (i) gM8 aggregated following heat treatment; (ii) gM8 was a virion component; (iii) gM8 and gN8 were N-glycosylated; (iv) gM8 formed a specific complex with gN8; and (v) gN8 was required for functional processing of gM8. Co-expression of gM8 and gN8 inhibited cell fusion induced either by a combination of herpes simplex virus type 1 glycoproteins or by Molony murine leukaemia virus envelope protein. These results indicate that, in addition to the similar biochemical properties, the fusion inhibition reported previously only for alphaherpesviruses is a function conserved in the gammaherpesvirus subfamily.Herpesviruses encode several glycoproteins; some are unique to a particular subfamily or virus, while others, including glycoprotein B (gB), gH, gL, gM and gN, are conserved throughout the herpesvirus family. These glycoproteins are likely to play essential and similar roles during the infectious cycle. However, their functional similarities cannot be assumed based only on the conservation of sequences and biochemical characteristics. An instructive example in this regard is gB, which was not detectable in the virions of some gammaherpesviruses (Gong & Kieff, 1990; Stewart et al., 1994). Although gMs and gNs of most herpesviruses share several biochemical characteristics in addition to their sequence homologies (Baines & Roizman, 1993;Dijkstra et al., 1996; Jöns et al., 1998;Lake et al., 1998;Mach et al., 2000;MacLean et al., 1993;Osterrieder et al., 1996;Pilling et al., 1994; Wu et al., 1998), differences have been noted in their fundamental properties, specifically, the degree of dispensability for growth in cell culture and effects on penetration and/or egress (Adams et al., 1998;Baines & Roizman, 1991;Dijkstra et al., 1996;Hobom et al., 2000; Jöns et al., 1998; König et al., 2002; Lake & HuttFletcher, 2000;MacLean et al., 1991;Osterrieder et al., 1996; Tischer et al., 2002). Because the properties of these glycoproteins have mainly been studied in alphaherpesviruses, additional studies are required to clarify whether any function found in alphaherpesviruses is applicable to other subfamilies. Neither the gM nor the gN homologue of human herpesvirus 8 (HHV-8) has yet been studied in detail. Therefore, in this study, we analysed their biochemical and functional properties.Expression of HHV-8 gM (gM8) (ORF39) was analysed by immunoblotting of cell lysates prepared from BCBL-1 cells, a primary effusion lymphoma cell line containing latent HHV-8 (Renne et al., 1996). The lytic infection was induced by treatment of the cells with 20 ng 12-Otetradecanoylphorbol 13-acetate (TPA) ml 21. Anti-gM8 serum was prepared by immunization of rabbits with...