No abstract

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-{4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl}-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.The herpesvirus family has many members that are human pathogens and make a significant contribution to morbidity and mortality associated with viral diseases. Based on criteria such as host cell specificity, oncogenicity, length of replication cycle, and genome arrangement, the herpesviruses have been divided into alpha-, beta-, and gammaherpesviruses (31). The alphaherpesviruses herpes simplex virus (HSV) types 1 and 2 latently infect nerve cells. HSV-1 is primarily associated with herpes labialis, and HSV-2 is associated with herpes genitalis, but both types have been associated with both diseases (28,39,47). In immunocompetent adults, these diseases often recur due to reactivation of the virus from the latent state. HSV infections of immunocompromised patients such as transplant and AIDS patients are often chronic and fatal. Current therapy for HSV disease consists of nucleoside analogs such as acyclovir (ACV) and valacyclovir, a prodrug of ACV, and pencyclovir (PCV) and its prodrug, famcyclovir. ACV and PCV are selectively phosphorylated by the viral thymidine kinase in HSVinfected cells, followed by further phosphorylation to the triphosphate by cellular kinases. Triphosphorylated ACV and PCV are both inhibitors of the viral DNA polymerase, and ACV also acts as a chain terminator when incorporated into the nascent viral DNA chain (4, 13). Drug resistance can occur in chronic infections, where replication is ineffectively curtailed by the immune system. Recently increasing numbers of drugresistant HSV strains have been isolated from immunocompromised people. The mechanism of resistance of most ACV-resistant isolates is...

Glvr1 encodes the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related gene Glvr2 encodes the human receptor for amphotropic murine leukemia viruses (A-MLVs). The two proteins are 62% identical in their amino acid sequences and are predicted to have 10 transmembrane domains and five extracellular loops. A stretch of nine amino acids (region A) in the predicted fourth extracellular loop was previously shown to be critical for the function of Glvr1 as receptor for GALV and FeLV-B. Glvr1 and -2 show clusters of amino acid differences in several of their predicted extracellular loops, with the highest degree of divergence in region A. Chimeras were made between the two genes to further investigate the role of Glvr1 region A in defining receptor specificity for GALV and FeLV-B and to map which regions of Glvr2 control receptor specificity for A-MLVs. Region A fromGlvr1 was sufficient to confer receptor specificity for GALV upon Glvr2, with the same chimera failing to act as a receptor for FeLV-B.

The expression of the human cytomegalovirus (HCMV) UL97 open reading frame in infected or transfected cells in the presence of the antiherpes compound ganciclovir (GCV) results in the intracellular phosphorylation of GCV. There are conventional kinase domains within the UL97-encoded protein (pUL97). However, the role of pUL97 in the HCMV replication cycle, and the mechanism by which it causes phosphorylation of GCV, are currently unknown. Herein, the biosynthesis and biogenesis of pUL97 was studied in HCMV-infected cells. pUL97 is expressed with early-late kinetics and is posttranslationally modified by phosphorylation. This phosphorylation occurs within 1 hr after synthesis, affects the electrophoretic mobility of pUL97, and is independent of the presence of other HCMV proteins. pUL97 was localized to the nucleus of infected cells and found in the HCMV virions. Thus, pUL97 is a virion phosphoprotein, and a likely tegument component.

Expression of human GLVR1 in mouse cells confers susceptibility to infection by gibbon ape leukemia virus (GALV), while the normally expressed mouse Glvr-1 does not. Since human and murine GLVR1 proteins differ at 64 positions in their sequences, some of the residues differing between the two proteins are critical for infection. To identify these, a series of hybrids and in vitro-constructed mutants were tested for the ability to confer susceptibility to infection. The results indicated that human GLVR1 residues 550 to 551, located in a cluster of seven of the sites that differ between the human and mouse proteins, are the only residues differing between the two which must be in the human protein form to allow infection. Sequencing of a portion of GLVR1 from the rat (which is infectible) confirmed the importance of this cluster in that it contained the only notable differences between the rat and mouse proteins. This region, which also differs substantially between the rat and the human proteins, therefore exhibits a pronounced tendency for polymorphism.