Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells

Lanford, Robert E.; Notvall, Lena; Beames, Burton

doi:10.1128/jvi.69.7.4431-4439.1995

Cited by 114 publications

(79 citation statements)

References 57 publications

(86 reference statements)

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“…HBV polymerases were purified using an affinity column containing the anti-FLAG M2 monoclonal antibody as described. 43 Briefly, the baculovirus-infected Sf21 cell pellet was extracted with 20 mL of phosphate-buffered saline containing 10% glycerol, 0.5% NP-40, protease inhibitors (100 µmol/L leupetin, 1 mmol/L pefabloc, 10 µmol/L aprotinin, 1 µg/mL pepstatin, and 1 mmol/L ethylenediaminetetraacetic acid), 50 U/mL RNasin (Promega). The lysate was cleared by centrifugation at 15,000g for 10 minutes.…”

Section: Methodsmentioning

confidence: 99%

In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

et al. 2000

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Several mutations (V521L, P525L, L528M, T532S, and V555I) in the gene for hepatitis B virus (HBV) polymerase have been identified in HBV isolated from patients that displayed break-through viremia during famciclovir treatment. To determine whether these mutations cause phenotypic resistance to famciclovir, we compared the inhibition constants (K i ) of penciclovir triphosphate (PCVTP, the active metabolite of famciclovir) for recombinant wild-type and mutant HBV polymerases containing these mutations. In in vitro enzymatic assays, the V555I mutation displayed the most resistance (with K i increased by 6.2-fold) to PCVTP. The V521L and L528M mutations showed moderately decreased sensitivity to PCVTP (K i increased by G3-fold). We also analyzed the cross-resistance profiles of these variants for adefovir and lamivudine, two other antiviral agents that also inhibit DNA replication by HBV polymerase. All 5 famciclovir-associated mutations were sensitive to adefovir diphosphate (ADVDP) in in vitro enzymatic assays (F2.3-fold decreased sensitivity). The V521L, L528M, and T532S mutations were also sensitive to lamivudine triphosphate (LAMTP); however, the P525L and V555I mutations displayed moderately decreased sensitivity to LAMTP in enzymatic assays (3.6-fold decreased sensitivity). The lamivudine-resistant mutations M552I, M552V, and L528M؉M552V, which were previously shown to display 8-to 25-fold resistance to LAMTP, were less resistant (I3.1-fold) to PCVTP. (HEPATOLOGY 2000;31:219-224.)Hepatitis B viral infection is a major cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and it is one of the 10 most common causes of death worldwide. 1 Current treatment regimens rely primarily on interferon alfa therapy. However, interferon has limited efficacy, is expensive, requires parenteral administration, and is associated with frequent and unpleasant side effects. Nucleoside or nucleotide analogs, which inhibit DNA replication through hepatitis B virus (HBV) polymerase, represent a new class of promising anti-HBV therapeutics. In addition to lamivudine, 2-9 which was approved recently for the treatment of hepatitis B infection in the United States, several nucleoside and nucleotide analogs including famciclovir 5,10,11 and adefovir dipivoxil 12-14 are currently being evaluated in late stage clinical trials. These compounds have been shown to be well tolerated and can produce rapid and marked decreases in serum HBV-DNA levels.Famciclovir is the oral prodrug of penciclovir, which has to be converted to penciclovir triphosphate by cellular enzymes to become an active inhibitor against HBV polymerase by competing with the natural substrate deoxyguanosine triphosphate (dGTP). 15 Famciclovir was originally approved for the treatment of herpes simplex virus and herpes zoster virus. 16,17 Later, famciclovir was shown to have activity against human and duck hepatitis B viruses in enzymatic and cell culture assays and in animal models of infection. 15,[18][19][20][21] Clinical studies subsequently showed...

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Section: Methodsmentioning

confidence: 99%

In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

et al. 2000

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“…Furthermore, purine nucleoside analogues can potentially inhibit the priming activity of the polymerase since the nucleotides present in the primed sequence of HBV minus-strand DNA are 5Ј-GAA-3Ј. [11][12][13]28 As shown in Table 1, a number of nucleoside/nucleotide analogues are being tested for use in CH-B. Except for entecavir, LY582563, and MIV-210, these compounds are in the unnatural "L" conformation.…”

Section: Small Molecule Inhibitorsmentioning

confidence: 99%

New targets and possible new therapeutic approaches in the chemotherapy of chronic hepatitis B

2003

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“…When trans-acting POL is produced in vivo, it is generated from a mRNA containing ε in cis [5,7,18,24,30,35,41]. The activity of POL expressed in yeast cells [37] and in insect cells [42] is also strictly dependent on the presence of a copy of ε in cis. Although Ziermann & Ganem [16] showed that ε is not required in cis for the packaging activity of POL, they could not exclude the possibility that ε might be required in cis to activate replication functions of POL in vivo.…”

Section: Activation Of Polymerase and Template Restrictionmentioning

confidence: 99%

“…For DHBV POL, the priming reaction requires dGTP, but when the C residue in the bulge is mutated to G the priming reaction requires dCTP [52]. Extrapolating the mechanism of priming from DHBV to HBV, Lanford et al [42] suggested that the preference for priming with a T residue may result from priming at the A residue in the sequence ACUU found in the bulge and in DR1. Using DNA transfection of complete HBV genomes, Nassal & Rieger [53] demonstrated that the 3′ half of the bulge of ε serves as the template for the DNA primer, whereas its 5′ adjacent nucleotides, apparently in closer contact with POL [54], are likely to have a role in arresting primer elongation because they are not accessible for polymerization [55].…”

Section: Initiation Of Reverse Transcriptionmentioning

confidence: 99%

Structure and function of the encapsidation signal of hepadnaviridae

Kramvis

Kew

1998

Journal of Viral Hepatitis

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The hepatitis B virus (HBV) and other members of the hepadnaviridae replicate by reverse transcription of an RNA intermediate, pregenomic RNA (pgRNA). pgRNA is also translated into core protein and polymerase (reverse transcriptase) protein. Before being reverse transcribed, pgRNA is sequestrated from the cytoplasm by being packaged, together with polymerase, into subviral particles composed of core protein. For pgRNA to be encapsidated, its 5' end is folded into a stem-loop structure, known as the encapsidation signal or epsilon (epsilon). This stable bipartite stem-loop structure contains a bulge and an apical loop. Besides encapsidation, epsilon is involved in the activation of polymerase, in template restriction and in the initiation of DNA synthesis by reverse transcription. HBV DNA encoding epsilon forms part of the template that is translated into the precore/core fusion protein that is in turn post-translationally modified to produce hepatitis B e antigen (HBeAg). The DNA encoding epsilon may be recombinogenic. Mutations within epsilon can affect its function and sequence conservation within epsilon in natural isolates is therefore high. epsilon could provide a practical target for antiviral therapy.

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Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells

Cited by 114 publications

References 57 publications

In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

New targets and possible new therapeutic approaches in the chemotherapy of chronic hepatitis B

Structure and function of the encapsidation signal of hepadnaviridae

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