Activity and mechanism of action of HDVD, a novel pyrimidine nucleoside derivative with high levels of selectivity and potency against gammaherpesviruses.

A novel nucleoside analogue, 1-[(2S,4S-2-(hydroxymethyl)-1,3-dioxolan-4-yl]5-vinylpyrimidine-2,4(1H,3H)-dione, or HDVD, was evaluated against a wide variety of herpesviruses and was found to be a highly selective inhibitor of replication of the gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). HDVD had also a pronounced inhibitory activity against murine herpesvirus 68 (MHV-68) and herpes simplex virus 1 (HSV-1). In contrast, replication of herpesvirus saimiri (HVS), HSV-2, and varicella-zoster virus (VZV) was weakly inhibited by the compound, and no antiviral activity was determined against human cytomegalovirus (HCMV) and rhesus rhadinovirus (RRV). The HDVD-resistant virus phenotype contained point mutations in the viral thymidine kinase (TK) of HSV-1, MHV-68, and HVS isolates. These mutations conferred cross-resistance to other TK-dependent drugs, with the exception of an MHV-68 mutant (E358D) that exhibited resistance only to HDVD. HSV-1 and HVS TK-mutants isolated under selective pressure with bromovinyldeoxyuridine (BVDU) also showed reduced sensitivity to HDVD. Oral treatment with HDVD and BVDU was assessed in an intranasal model of MHV-68 infection in BALB/c mice. In contrast to BVDU treatment, HDVD-treated animals showed a reduction in viral DNA loads and diminished viral gene expression during acute viral replication in the lungs in comparison to levels in untreated controls. The valyl ester prodrug of HDVD (USS-02-71-44) suppressed the latent infection in the spleen to a greater extent than HDVD. In the present study, HDVD emerged as a highly potent antiviral with a unique spectrum of activity against herpesviruses, in particular, gammaherpesviruses, and may be of interest in the treatment of virus-associated diseases.

Evidence for an important role of serine 16 and its phosphorylation in the stabilization of c-Mos.

The c-Mos serine/threonine protein kinase is an essential component of cytostatic factor (CSF), which is required for metaphase II arrest of eggs in vertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylated in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein. Here we provide evidence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To investigate the role of this phosphorylation, Ser-16 was substituted with alanine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mouse c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos. Constructs expressing mutant versions of Mos were transfected into COS-1 and NIH3T3 cells in a transient and stable manner, respectively. Synthesis and proteolysis of Mos were evaluated by pulse-chase analysis of 35S-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16A mutation but not the S16E mutation inhibited Mos interaction with a cellular protein, p35, suggesting that phosphorylation at Ser-16 may promote Mos interaction with p35. As expected from its destabilizing effect, the S16A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably transfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the second residue from the amino-terminus. In contrast to Mos, neither the stability nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 becomes Pro-33) was affected by the S16A mutation. To provide further proof that, similar to c-Mos, the S16A mutant is recognized by the proteolysis system through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an important role in the regulation of c-Mos and that phosphorylation at Ser-16 may inhibit proteolysis of c-Mos.

Evidence of an interaction between Mos and Hsp70: a role of the Mos residue serine 3 in mediating Hsp70 association.

c-Mos is a germ cell-specific MAP kinase kinase kinase (MAPKKK) that plays an essential role during meiotic divisions of oocytes. c-Mos is a key component of an activity, cytostatic factor, required for metaphase II arrest of unfertilized eggs in vertebrates. To understand the regulation of c-Mos, we are investigating c-Mos-interacting proteins. We provide evidence that mouse c-Mos binds to Hsp70, a molecular chaperone. Hsp70 was found to associate with Mos ectopically expressed in COS-1 cells. Mos-Hsp70 complexes could be immunoprecipitated with both Mos and Hsp70 antibodies. Despite a low-abundance of Mos, the Hsp70 antibody immunoprecipitated Mos as the major protein. Of importance, the Mos protein present in anti-Hsp70 immunoprecipitates functioned as an active MAPKKK indicating that it is not grossly misfolded. It is known that c-Mos protein kinase activity in cell extracts of transfected COS-1 or NIH3T3 cells is labile. We found that the inclusion of adenosine triphosphate (ATP) in cell extracts protected against the loss of Mos kinase activity. In the absence of ATP from cell extracts, protein kinase activity of Mos was lost within 6 h on ice even though the Mos protein was not degraded and remained bound to Hsp70. Based on our identification of c-Mos-Hsp70 interaction, one of the roles of ATP may be to assist the regulation of c-Mos via ATP involvement in the protein-folding function of Hsp70 and possibly other molecular chaperones. We also detected by coimmunoprecipitation a physical association between endogenous c-Mos and Hsp70 in Xenopus eggs. To provide further evidence for the functional significance of Hsp70 interaction to Mos function, we show that the residue serine 3 in Mos, which is important for the regulation of protein kinase activity of Mos is also important for Hsp70 association.

Evidence of a functional interaction between serine 3 and serine 25 Mos phosphorylation sites. A dominant inhibitory role of serine 25 phosphorylation on Mos protein kinase.

Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.