Inhibition Of Viral DNA Polymerase Research Articles

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.

Genome-wide gene expression analysis of anguillid herpesvirus 1

BackgroundWhereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR.ResultsFour immediate-early genes – open reading frames 1, 6A, 127 and 131 – were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins.ConclusionsOverall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.

Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

Cidofovir or (S)-HPMPC is one of the three antiviral drugs that might be used for the treatment of orthopoxvirus infections. (S)-HPMPC and its 2,6-diaminopurine counterpart, (S)-HPMPDAP, have been described to select, in vitro, for drug resistance mutations in the viral DNA polymerase (E9L) gene of vaccinia virus (VACV). Here, to extend our knowledge of drug resistance development among orthopoxviruses, we selected, in vitro, camelpox viruses (CMLV) resistant to (S)-HPMPDAP and identified a single amino acid change, T831I, and a double mutation, A314V+A684V, within E9L. The production of recombinant CMLV and VACV carrying these amino acid substitutions (T831I, A314V, or A314V+A684V) demonstrated clearly their involvement in conferring reduced sensitivity to viral DNA polymerase inhibitors, including (S)-HPMPDAP. Both CMLV and VACV harboring the A314V change showed comparable drug-susceptibility profiles to various antivirals and similar impairments in viral growth. In contrast, the single change T831I and the double change A314V+A684V in VACV were responsible for increased levels of drug resistance and for cross-resistance to viral DNA polymerase antivirals that were not observed with their CMLV counterparts. Each amino acid change accounted for an attenuated phenotype of VACV in vivo. Modeling of E9L suggested that the T→I change at position 831 might abolish hydrogen bonds between E9L and the DNA backbone and have a direct impact on the incorporation of the acyclic nucleoside phosphonates. Our findings demonstrate that drug-resistance development in two related orthopoxvirus species may impact drug-susceptibility profiles and viral fitness differently.

Human Cytomegalovirus UL44 Concentrates at the Periphery of Replication Compartments, the Site of Viral DNA Synthesis

The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In contrast, the viral single-stranded-DNA binding protein UL57 was distributed throughout replication compartments. Using "click chemistry" to detect 5-ethynyl-2'-deoxyuridine (EdU) incorporation into replicating viral DNA and pulse-chase protocols, we found that viral DNA synthesis occurs at the periphery of replication compartments and that replicated viral DNA subsequently localizes to the interior of replication compartments. The interiors of replication compartments also contain regions in which UL44 and EdU-labeled DNA are absent. The treatment of cells with a viral DNA polymerase inhibitor reversibly caused the dispersal of both UL44 and EdU-labeled viral DNA from replication compartments, indicating that ongoing viral DNA synthesis is necessary to maintain the organization of replication compartments. Our results reveal a previously unappreciated complexity of the organization of human cytomegalovirus replication compartments.

Herpèsvirus et herpèsviroses humaines

Les herpèsvirus sont des virus largement répandus dans la biosphère et infectant un très grand nombre d’espèces animales différentes. Ils appartiennent tous à l’ordre des Herpesvirales et aux trois familles Herpesviridae, Alloherpesviridae, et Malacoherpesviridae. Ils partagent de nombreuses propriétés communes : une structure de virus à ADN enveloppé de grande taille, une capacité à persister indéfiniment de façon silencieuse chez leur hôte après la primo-infection et à se réactiver ensuite pour donner à nouveau une infection lytique, l’établissement d’un équilibre vis-à-vis des mécanismes de défense de l’hôte, et la gravité des symptômes quand ces défenses sont amoindries. Les huit herpèsvirus humains sont les virus herpes simplex de type 1 et 2 (HSV-1, HSV-2), le virus de la varicelle et du zona (VZV), le cytomégalovirus (CMV), le virus Epstein-Barr (EBV) et les herpèsvirus humains HHV-6, -7 et -8, ce dernier étant encore appelé herpèsvirus associé à la maladie de Kaposi (KSHV). Le tropisme cellulaire, qu’il s’agisse de la multiplication active ou de l’état de latence, varie selon l’espèce virale et détermine en partie le pouvoir pathogène de ces virus qui ont un comportement opportuniste. Le diagnostic des infections aiguës repose actuellement essentiellement sur la PCR, en particulier les techniques en temps réel permettant un quantification virale. La prévention des infections humaines par la vaccination se limite au VZV. La chimiothérapie antivirale, fondée sur des inhibiteurs de l’ADN polymérase des herpèsvirus, permet de traiter les infections aiguës mais elle n’est pas efficace sur l’infection latente ou en cas de résistance consécutive à la sélection de mutations du génome viral.

Efficient Inhibition of Human Cytomegalovirus DNA Polymerase Expression by Small Hairpin RNA In Vitro

The aim of this study is to try to develop an RNA interference (RNAi) approach to human cytomegalovirus (HCMV) therapy by inhibition of viral DNA polymerase (product of the UL54 gene) expression in vitro. The fusion protein expression plasmid pEGFP-UL54 was constructed and cotransfected into AD293 cells with the lentiviral vector pGCL-GFP expressing small hairpin RNA (shRNA) especially targeting the UL54 gene. At 24, 48, and 72 h posttransfection, expression of the fusion protein was detected by fluorescence microscopy and Western blot. The level of UL54 mRNA was semiquantitated by reverse transcription polymerase chain reaction. It was found that, compared with the negative control, expression of the EGFP-UL54 fusion protein was efficiently inhibited and the mRNA level of the UL54 gene decreased significantly in cells introduced with a shRNA producing plasmid called pGCL-UL54-1479. However, the plasmid pGCL-UL54-2884 had no significant inhibitive effect on protein expression or mRNA level. It may be concluded that introduction of shRNA targeting UL54 gene of HCMV could specifically inhibit the expression of viral DNA polymerase in vitro. Nucleotides 1479-1499 of the UL54 gene seems to be an effective target site of RNAi.

Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

Targeting Enzymes with Phosphonate-Based Inhibitors: Mimics of Tetrahedral Transition States and Stable Isosteric Analogues of Phosphates

Phosphorus plays a fundamental role in cell since it is a part of many biomolecules and biological metabolites which include phospholipids, nucleic acids, proteins, polysaccharides or nucleotide cofactors. Phosphorus is most commonly found under its highest oxidized state as in orthophosphate or phosphate esters. However, there are also examples of naturally occurring molecules bearing phosphorus atoms at lower oxidized state that contain one carbon to phosphorus (P-C) bond. These compounds, so-called phosphonates, are much more resistant to chemical and enzymatic hydrolysis, thermal decomposition and photolysis than phosphates. The first example of a natural phosphonate, 2-aminoethylphosphonic acid (2-AEP), was reported in 1959 and shown to be a constituent of lipids, proteins and polysaccharides. Since the beginning of the 20th century, with the discovery of the Arbuzov reaction, synthetic phosphonates have been developed which have applications as therapeutic agents (e.g. antibiotics, anti-viral, or trypanocidal drugs) as well as insecticides and herbicides. Recently, phosphonate derivatives have been extensively used as enzyme inhibitors due to their specific structural and electronic properties. First, since they differ from phosphates by the single substitution of one oxygen atom with a carbon atom, phosphonates have been widely used as isosteric mimics of phosphates in the design of analogues of enzyme substrates or cofactors. They offer the advantage of a much greater stability to hydrolysis and resistance to proteases than phosphates and therefore can be used to mimic highly chemically unstable phosphates like carboxy-phosphates. Such a strategy was shown successful for generating competitive inhibitors of glycolytic enzymes (triose/hexose phosphate analogues), phosphatases or viral DNA polymerases (phosphonate nucleotides), with applications as chemotherapeutic agents against trypanosomiasis, hepatitis B or human immunodeficiency virus. Moreover, introduction of one or two fluorine atoms on the α-methylene group was also used to provide a better mimic of phosphate geometric and electronic properties by lowering the pKa of the phosphonate and increasing its P-Cα-Cβ dihedral angle. Phosphonic acids have also been extensively studied as enzyme inhibitors mimicking tetrahedral transition states. They proved to be potent competitive inhibitors of peptidases as mimics of the tetrahedral gem-diolate transition state of peptide bond hydrolysis, and of lipases, as mimics of configuration and charge distribution of the first transition state in triglyceride hydrolysis. This chapter summarizes the most recent studies on phosphonate-based enzyme inhibitors with special emphasis on isosteric analogues of phosphorylated substrates and mimics of tetrahedral transition states for peptide bond hydrolysis.

Antiviral therapeutic efficacy of foscarnet in hepatitis B virus infection

Foscarnet (PFA), a viral DNA polymerase inhibitor, is a clinical agent for herpes viruses. The goal of the study was to evaluate the therapeutic efficacy of PFA in hepatitis B virus (HBV) infection. Intravenous infusion of PFA (1 g/day) for 4 weeks significantly reduced serum HBeAg ( p < 0.01) and HBV DNA copies ( p < 0.05) in 31 patients who were diagnosed with active chronic HBV infection (CHB) and had not received antiviral treatment previously. Alanine aminotrans-aminase (ALT), aspartate aminotransaminase (AST) and gamma glutamyl transpeptidase (γ-GT) of the patients declined ( p < 0.001, 0.001 and 0.01, respectively). Kidney function (blood creatinine and urea nitrogen) remained unchanged. Another 21 lamivudine-resistant CHB patients with mutations at the tyrosine–methionine–aspartate–aspartate motif (YMDD) displayed a response to PFA similar to that mentioned above, with reductions in HBeAg ( p < 0.05), HBV DNA ( p < 0.01) and liver enzymes (ALT and AST, p < 0.001; γ-GT, p < 0.05). Moreover, PFA reduced serum HBeAg ( p < 0.01), HBV DNA ( P < 0.05), AST ( p < 0.05) and ALT ( p < 0.02) in a cohort of 13 severe CHB patients with advanced liver damage. PFA was also evaluated in vitro and in vivo. PFA inhibited HBV DNA replication in HBV-transfected human HepG2 cells (2.2.15 cells) with reduced amount of HBV RC-DNA and DS-DNA. In the duck HBV-infected ducklings, PFA reduced viral DNA and duck HBsAg in the serum ( p < 0.01 for both).

Impact of 2-Bromo-5,6-Dichloro-1-β- d -Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.

Brivudin (bromovinyl deoxyuridine).

Brivudin is an oral thymidine analogue indicated for the early treatment of acute herpes zoster in immunocompetent adults. It has high, selective activity against varicella zoster virus (VZV), inhibiting VZV replication, possibly through competitive inhibition of viral DNA polymerase, or by acting as an alternative substrate to deoxythymidine triphosphate, causing viral DNA strand breakage. In a large, 7-day, phase III trial in immunocompetent patients with herpes zoster, once-daily brivudin 125mg was significantly more effective than oral acyclovir 800mg five times daily in reducing the mean time from start of treatment to last vesicular eruption, and was as effective as acyclovir at healing lesions and alleviating acute zoster-related pain. The likelihood of developing post-herpetic neuralgia (PHN) in immunocompetent patients aged > or =50 years was significantly lower with brivudin than with acyclovir. Brivudin was as effective as oral famciclovir 250mg three times daily in terms of the prevalence of PHN, the time to last vesicular eruption and lesion healing in another large, 7-day, phase III study in immunocompetent patients with herpes zoster. Oral brivudin is generally well tolerated, with a similar tolerability profile to those of oral acyclovir or famciclovir. Nausea was the most commonly reported adverse event.

Prolonged survival in AIDS-related progressive multifocal leucoencephalopathy following anti-retroviral therapy and cidofovir

We describe the case of a 44-year-old man who developed sub-acute cerebellar ataxia due to AIDS-related progressive multifocal leucoencephalopathy (PML) caused by JC virus. Following treatment with highly active anti-retroviral therapy (HAART) and cidofovir, he made a marked neurological improvement and is leading an independent life 18 months after the diagnosis of PML. Early recognition of AIDS-related PML and treatment with HAART improves prognosis. Cidofovir, an inhibitor of viral DNA polymerase, appears to have an additive beneficial effect and should be considered especially in patients who fail to improve despite treatment with HAART and in patients who have a high JC virus load in CSF.

Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a unique mode of action.

Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-beta-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-beta-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC(50)) of 0.12 +/- 0.01 microM compared to a mean IC(50) for GCV of 0.53 +/- 0.04 microM, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole D-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of L-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.

Novel non-nucleoside inhibitors of cytomegaloviruses (BAY 38-4766): in vitro and in vivo antiviral activity and mechanism of action.

For two decades it has been impossible to develop drugs with novel mechanisms of action against herpesviruses, and treatment has been confined largely to the use of inhibitors of viral DNA polymerase. As a representative of a novel inhibitory approach, the non-nucleosidic BAY 38-4766 was identified as a highly selective inhibitor of human cytomegalovirus (HCMV). The compound selectively inhibits not only HCMV strains, including ganciclovir-resistant, ganciclovir/foscarnet and ganciclovir/cidofovir double-resistant clinical isolates, but also a number of monkey and rodent cytomegaloviruses. In a murine cytomegalovirus (MCMV) pathogenicity model in mice, antiviral efficacy and excellent tolerability were demonstrated. BAY 38-4766-resistant HCMV and MCMV strains are not cross-resistant to the nucleoside analogues ganciclovir and cidofovir or the pyrophosphate analogue foscarnet, indicating a different mode of action. Mechanistic studies demonstrated that the high selectivity of this drug class is most likely due to the inhibition of a late stage of the viral replication cycle. Sequence analyses of resistant HCMV and MCMV strains revealed mutations in UL89 and UL104, proteins known to be involved in viral DNA cleavage and packaging. Consequently, the drug is highly specific for the viral as opposed to cellular functions, since UL89 is related to a bacteriophage terminase and no human equivalent exists. In addition, because some of the genes of the viral DNA cleavage and packaging complex are highly conserved among herpesviruses, development of broad-spectrum agents covering additional human herpesviruses might be possible using this approach.

Tolerability of treatments for viral hepatitis.

Interferon-alpha is the most widely used antiviral drug in chronic hepatitis B and C. Tolerability is usually good and serious adverse effects are rare. Most of the adverse effects are mild or transient and do not necessitate drug withdrawal. More than 90% of patients who are given interferon-alpha achieve 6 months to 1 year of treatment without serious adverse effects. The serious adverse effects usually occur in predisposed patients with pre-existing organ dysfunction. Nevertheless, careful selection of patients for therapy and observation during therapy are recommended. Nucleoside analogues are promising drugs in the treatment of chronic hepatitis B through inhibition of viral DNA polymerase. Lamivudine has been licensed for use in this indication. Its tolerability is excellent even when used for periods of 1 year or more. The main concern is the relatively high incidence of viral resistance resulting in breakthrough during or relapse after therapy. In the treatment of chronic hepatitis C, ribavirin, in combination with interferon-alpha is currently the reference therapy. The main adverse effect is haemolytic anaemia, which necessitates careful monitoring and adjustment of dosage in many cases. Recently, large trials showed the better efficacy of pegylated interferons as compared with standard interferon. The combination of pegylated interferon with ribavirin is under evaluation.

Kaposi's sarcoma-associated herpesvirus open reading frame 50/Rta protein activates the entire viral lytic cycle in the HH-B2 primary effusion lymphoma cell line.

Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.

The BFRF1 gene of Epstein-Barr virus encodes a novel protein.

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs.

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.

Clinical pharmacokinetics of the antiviral nucleotide analogues cidofovir and adefovir.

Cidofovir and adefovir are members of a new class of antiviral compounds. They are acyclic phosphonate analogues of deoxynucleoside monophosphates. Both compounds undergo intracellular activation to form diphosphates that are potent inhibitors of viral DNA polymerases. Cidofovir has broad spectrum antiviral activity against herpesviruses, papillomaviruses and poxviruses, whereas adefovir has potent activity against retroviruses and certain DNA viruses, including herpesviruses and hepadnaviruses. Intravenous cidofovir is approved for treatment of cytomegalovirus retinitis in patients with AIDS. Cidofovir and adefovir are dianionic at physiological pH and have low oral bioavailability in animals and humans. After intravenous administration to HIV-infected patients, the pharmacokinetics of both drugs are independent of dose and are consistent with preclinical data. Systemic exposure is proportional to the intravenous dose and both drugs are cleared by the kidney and excreted extensively as unchanged drug in the urine. Intracellular activation of a small fraction (< 10%) of the dose by cellular kinases leads to prolonged antiviral effects that are not easily predicted from conventional pharmacokinetic studies. The observed rate of elimination of cidofovir and adefovir from serum may not reflect the true duration of action of these drugs, since the antiviral effect is dependent on concentrations of the active phosphorylated metabolites that are present within cells. For both drugs, > 90% of an intravenous dose is recovered unchanged in the urine over 24 hours. Metabolism does not contribute significantly to the total clearance of either drug. Concomitant oral probenecid decreases both the renal clearance of cidofovir and the incidence of nephrotoxicity, presumably by blocking its active tubular secretion. This is the basis of the clinical use of concomitant probenecid as a nephroprotectant during cidofovir therapy. Subcutaneous administration produces exposure equivalent to that following intravenous administration. Drug interaction studies with cidofovir are ongoing, but there is no evidence of an interaction between zidovudine and either cidofovir or adefovir. Clearance of cidofovir in patients with renal impairment showed a linear relationship to creatinine clearance. The low oral bioavailability of adefovir has led to the development of an oral prodrug, adefovir dipivoxil, currently in development for the treatment of HIV and hepatitis B infections.

Viral Glycoproteins Accumulate in Newly Formed Annulate Lamellae following Infection of Lymphoid Cells by Human Herpesvirus 6

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absent in uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no AL were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was further confirmed by experiments using inactivated virus or performed in presence of the viral DNA polymerase inhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracture labeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmic reticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with an anti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes and endoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteins selectively accumulate in AL. In addition, gold labeling with Helix pomatia lectin and wheat germ agglutinin indicated that AL cisternae, similar to cis-Golgi membranes, contain intermediate, but not terminal, forms of glycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viral glycoprotein storage compartment and as a putative site of O-glycosylation.