Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinasedependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding motif or the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.All the human herpesviruses encode well-conserved serine/ threonine protein kinases that are important in viral infection (51) and are thought to phosphorylate substrates that are also targets of cdc2 (33). Herpes simplex virus (HSV) UL13 and Epstein-Barr virus BGLF4 phosphorylate eukaryotic elongation factor 1delta (34), and HSV UL13 and human cytomegalovirus (HCMV) pUL97 both phosphorylate the carboxylterminal domain of RNA polymerase II (4, 12). Many other interesting activities of cellular proteins have previously been described, such as the activation of cdc2 by HSV UL13 (1) as well as the inhibition of histone acetylation (57) and activation of protein kinase A (5, 43) by HSV US3. Viral proteins can also be substrates for these kinases; the DNA polymerase processivity factors are substrates, and it appears to be a common theme in the herpesviruses (21,27,39,47). Studies examining the function of these kinases suggest that they are not strictly required for viral infection; however, they perform important functions that are required for replication in vivo and suggest that the effects of these kinases on host and viral targets are important (53,54,62).The UL97 protein kinase in HCMV is particularly important because of its relevance to antiviral therapy. This enzyme phosphorylates and activates the antiviral drug ganciclovir, which is the treatment of choice for HCMV infections (44, 63). Although this drug...

Recombinant human cytomegaloviruses that do not express UL97 kinase activity exhibit a distinctive plaque morphology characterized by the formation of highly refractile bodies late in infection. These structures were also observed in infected cells treated with the UL97 kinase inhibitor maribavir. Nuclear inclusions were purified to near homogeneity, and the constituent proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This analysis demonstrated that the aggregates were formed principally of the tegument proteins pp65 and ppUL25 but also contained additional virion structural proteins including the major capsid protein. Immunoblotting experiments confirmed these results and identified a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appeared to be dependent on pp65, since it was not induced in cells infected with a recombinant virus with this open reading frame deleted. Morphologically similar aggregates could be reproduced in nuclei of uninfected cells by overexpressing pp65, and their formation was prevented by coexpressing the UL97 kinase. Inhibition of UL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase abolished its ability to prevent aggregate formation. These data taken together suggest that the UL97 kinase impacts the aggregation of pp65 in the nuclei of infected cells. We propose that the kinase plays an important role in the acquisition of tegument during virion morphogenesis in the nucleus and that this activity represents an important step in the production of mature virus particles.

No abstract

Cidofovir (CDV) is an effective therapy for certain human cytomegalovirus (HCMV) infections in immuno-compromised patients that are resistant to other antiviral drugs, but the compound is not active orally. To improve oral bioavailability, a series of lipid analogs of CDV and cyclic CDV (cCDV), including hexadecyloxypropyl-CDV and -cCDV and octadecyloxyethyl-CDV and -cCDV, were synthesized and found to have multiple-log-unit enhanced activity against HCMV in vitro. On the basis of the activity observed with these analogs, additional lipid esters were synthesized and evaluated for their activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus, murine cytomegalovirus, varicella-zoster virus (VZV), EpsteinBarr virus (EBV), human herpesvirus 6 (HHV-6), and HHV-8. Using several different in vitro assays, concentrations of drug as low as 0.001 M reduced herpesvirus replication by 50% (EC 50 ) with the CDV analogs, whereas the cCDV compounds were generally less active. In most of the assays performed, the EC 50 values of the lipid esters were at least 100-fold lower than the EC 50 values for unmodified CDV or cCDV. The lipid analogs were also active against isolates that were resistant to CDV, ganciclovir, or foscarnet. These results indicate that the lipid ester analogs are considerably more active than CDV itself against HSV, VZV, CMV, EBV, HHV-6, and HHV-8 in vitro, suggesting that they may have potential for the treatment of infections caused by a variety of herpesviruses.

A series of 4-thionucleosides were synthesized and evaluated for activities against orthopoxviruses and herpesviruses. We reported previously that one analog, 5-iodo-4-thio-2-deoxyuridine (4-thioIDU), exhibits good activity both in vitro and in vivo against two orthopoxviruses. This compound also has good activity in cell culture against many of the herpesviruses. It inhibited the replication of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus with 50% effective concentrations (EC 50 s) of 0.1, 0.5, and 2 M, respectively. It also inhibited the replication of human cytomegalovirus (HCMV) with an EC 50 of 5.9 M but did not selectively inhibit Epstein-Barr virus, human herpesvirus 6, or human herpesvirus 8. While acyclovir-resistant strains of HSV-1 and HSV-2 were comparatively resistant to 4-thioIDU, it retained modest activity (EC 50 s of 4 to 12 M) against these strains. Some ganciclovir-resistant strains of HCMV also exhibited reduced susceptibilities to the compound, which appeared to be related to the specific mutations in the DNA polymerase, consistent with the observed incorporation of the compound into viral DNA. The activity of 4-thioIDU was also evaluated using mice infected intranasally with the MS strain of HSV-2. Although there was no decrease in final mortality rates, the mean length of survival after inoculation increased significantly (P < 0.05) for all animals receiving 4-thioIDU. The findings from the studies presented here suggest that 4-thioIDU is a good inhibitor of some herpesviruses, as well as orthopoxviruses, and this class of compounds warrants further study as a therapy for infections with these viruses.