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.

A series of nonnucleoside, N-␣-methylbenzyl-N-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.Varicella-zoster virus (VZV) causes a disseminated primary infection, chickenpox, and later may reactivate to cause herpes zoster, commonly known as shingles (3,26). Herpes zoster can result in pain lasting over 1 month from the onset of the rash, termed postherpetic neuralgia (PHN). PHN is often a debilitating condition that is quite difficult to treat (15,32,33).Acyclovir (ACV), valaciclovir, and famciclovir are the primary antivirals used for treating VZV infections in both immunocompetent and immunocompromised individuals (25). Unfortunately, despite these medications, PHN remains a major problem, and the currently available drugs have a limited effect on prevention of this condition. Thus, there is a need for additional, more effective medications to treat herpes zoster in adults.The development of antiviral resistance remains a concern, particularly when treatment relies on only a small number of approved drugs with common modes of action (13,14,25,26). ACV, valaciclovir (which is metabolized to acyclovir), and famciclovir (the prodrug of penciclovir) are all phosphorylated by the viral thymidine kinase as an initial step for intracellular activation. They are subsequently phosphorylated by cellular kinases, and the activated compounds inhibit the viral DNA polymerase. Sorivudine and brivudine are also potent inhibitors of VZV, and although they represent a different class of nucleoside analogues from ACV, they also require phosphorylation by the viral thymidine kinase for activation (18,31). While thymidine kinase-deficient VZV can be treated with foscarnet, a direct inhibitor of the viral DNA polymerase (25), this drug requires intravenous administration and has a marked potential for...

Hepatitis C virus (HCV) infection is the cause of significant long-term morbidity and mortality. While often asymptomatic, the majority of HCV infections result in chronic hepatitis that can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. It is estimated that about 170 million people worldwide, approximately 3% of the world's population, are infected with HCV (45). Presently, there is no specific antiviral agent directed against HCV and no vaccine for prevention of hepatitis C infection. The current approved treatments, interferon monotherapy or interferon in combination with ribavirin, have limited benefits. Interferon alone achieves a sustained viral response in only 10 to 20% of patients (32), while the recommended therapy of pegylated interferon in combination with ribavirin results in a sustained viral response in 54% of patients (23). The response rate is lower in patients who are infected with HCV genotype 1b (ϳ34%) (23,47). Adverse side effects, such as severe flu-like symptoms, depression, psychoses, and anemia, are associated with these treatments, causing approximately 20% of patients to discontinue therapy (13,17,33). Consequently, there is an urgent need for the development of an HCV-specific antiviral that is more effective, less toxic, and easier to administer than the present therapy.HCV, a member of the Flaviviridae family, is a positivesense, single-stranded RNA virus with a genome size of ϳ9.4 kb (26, 39). The genome RNA encodes a polyprotein of 3,010 to 3,011 amino acid residues in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein is processed by host and viral proteases (18,34). The nonstructural protein 5B (NS5B) is a virus-encoded RNA-dependent RNA polymerase (RdRp) that is responsible for replication of the viral RNA genome. A functional counterpart of NS5B does not exist in mammalian cells. For this reason, an inhibitor of NS5B could serve as an effective and selective agent for treating HCV infection.The enzymatic activity of the NS5B enzyme in vitro has been extensively characterized (9,19,21,40). Both the full-length and carboxyl-terminally truncated forms of NS5B have been shown to be functionally active in the presence of HCV or exogenous RNA templates. Although HCV replicates inefficiently in cell culture, viral replication, including the activity of the NS5B polymerase, can be studied in a cell culture system

SummaryWe have identi®ed the major endo-b-1,4-xylanase (XYN-1) in the aleurone of germinating barley grain, and show that it is expressed as a precursor of M r 61 500 with both N-and C-terminal propeptides. XYN-1 is synthesized as an inactive enzyme in the cytoplasm, and only becomes active at a late stage of germination when the aleurone ceases to secrete hydrolases. A series of processing steps, mediated in part by aleurone cysteine endoproteases, yields a mature active enzyme of M r 34 000. Processing and extracellular release of the mature enzyme coincide with the programmed cell death (PCD)-regulated disintegration of aleurone cells. We discuss the signi®cance of delayed aleurone cell-wall degradation by endoxylanases in relation to the secretory capacity of the aleurone, and propose a novel role for aleurone PCD in facilitating the export of hydrolases.