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John E Tavis

Publications and source records attributed to John E Tavis.

15 recordsLinked to original sources
PubMed2025

Discovery of bimodal hepatitis B virus ribonuclease H and capsid assembly inhibitors.

Hepatitis B virus (HBV) ribonuclease H (RNaseH) inhibitors are a potent class of antivirals that prevent degradation of the viral pregenomic RNA during reverse transcription and block formation of mature HBV DNAs. Development of HBV RNaseH inhibitors is entering advanced preclinical analyses. To ensure the mechanism of action was fully understood, we defined the effects of RNaseH inhibitors on other steps of HBV replication. Some N-hydroxypyridinedione (HPD) HBV RNaseH inhibitors significantly reduced accumulation of capsids in HBV-replicating cells. A representative HPD 1466, with a 50% effective concentration against HBV replication of 0.25 µM, decreased capsid and core protein accumulation by 50-90% in HepDES19 and HepG2.2.15 cells. Surprisingly, 1466 did not affect pregenomic RNA encapsidation, demonstrating a specific effect on empty capsids. HBV genomic replication was not necessary for 1466's inhibitory effect as it decreased capsid accumulation in cells transfected with replication-deficient mutants blocking pgRNA encapsidation (Δ-bulge), DNA synthesis (YMHA), and RNaseH (D702A) activities. 1466 also decreased capsid and core protein accumulation in cells transfected with a core protein expression plasmid, indicating that other HBV products are unneeded. 1466 reduced initial capsid assembly rates in biochemical assembly reactions employing purified core protein (Cp149), demonstrating a specific effect on HBV core protein. We conclude that the bimodal HPD HBV RNaseH inhibitor 1466 is the prototypic member of a new class of capsid assembly modulators (CAM) that inhibits capsid assembly rather than accelerating it, as all other CAM classes do. We propose that this class be called CAM-I, for CAM-inhibitor. These results lay the foundation for identifying bimodal HBV antivirals targeting the RNaseH and capsid assembly.

Hepatitis B virus↗
PubMed2007

Separation of near full-length hepatitis C virus quasispecies variants from a complex population.

A long RT-PCR (LRP) protocol was developed recently for robust amplification of a near full-length HCV genomic sequence from clinical samples, followed by efficient cloning [Fan, X., Xu, Y., Di Biceglie, A.M., 2006. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem. Biophys. Res. Commun. 346, 1163-1172]. In the present study, the LRP protocol has been estimated for its error rate and the validation by sequencing fully the near full-length HCV inserts from six recombinant clones derived from a patient sample with complex viral diversity. These sequences were compared with the near full-length HCV sequence that was generated by direct sequencing of multiple overlapped PCR products from the same sample, referred to as the population sequence. Comparative analysis confirmed the artificial nature of the PCR-assembled population sequence and identified potential domains for linked viral mutations. The data also suggested that the hypervariable region 1 (HVR1) may be a biological marker for the phenotype at the quasispecies level. These observations emphasize the significance of the use of near full-length genomic sequences for HCV genetic studies and for reverse genetic analysis using authentic quasispecies variants.

Cloning, Molecular↗
PubMed2006

The duck hepatitis B virus reverse transcriptase functions as a full-length monomer.

Hepadnaviral reverse transcription occurs within cytoplasmic capsid particles and is catalyzed by a virally encoded reverse transcriptase, but the primary structure and multimeric state of the polymerase during reverse transcription are poorly understood. We measured these parameters for the duck hepatitis B virus polymerase employing active enzyme translated in vitro and derived from intracellular core particles and mature virions. In vitro-translated polymerase immunoprecipitated as a monomer, and polymerase molecules with complementary defects in the enzymatic active site and tyrosine 96, which primes DNA synthesis, could not complement or inhibit each other in priming assays. Western analysis using antibodies recognizing epitopes throughout the polymerase combined with nuclease digestion of permeabilized virion-derived capsid particles revealed that only full-length polymerase molecules were in virions and that they were all covalently attached to large DNA molecules. Because DNA synthesis is primed by the polymerase itself and only one copy of the viral DNA is in each capsid, the polymerase must function as an uncleaved monomer. Therefore, a single polymerase monomer is encapsidated, primes DNA synthesis, synthesizes both DNA strands, and participates in the three-strand transfers of DNA synthesis, with all steps after DNA priming performed while the polymerase is covalently coupled to the product DNA. Because the N-terminal domain of the polymerase is displaced from the active site on the same molecule by the viral DNA during reverse transcription, P must be structurally dynamic during DNA synthesis. Therefore, non-nucleoside compounds that interfere with this change may be novel antiviral agents.

Animals↗
PubMed2006

Combining genetic and biochemical approaches to identify functional molecular contact points.

Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods.

Journal Article↗
PubMed2006

Suppression of mRNA accumulation by the duck hepatitis B virus reverse transcriptase.

Hepadnaviruses establish chronic liver infections, but the mechanisms of persistence and immune evasion are poorly understood. We previously found that the duck hepatitis B virus (DHBV) and hepatitis B virus reverse transcriptases (P protein) unexpectedly accumulate in the cytoplasm where they could affect function(s) beyond viral DNA synthesis, such as gene expression. Therefore, we measured effects of DHBV P on gene expression from reporter constructs and the viral genome. P reduced reporter expression at the mRNA level to approximately 30-40%, independent of reporter tested. Accumulation of the viral pregenomic RNA from its native promoter was suppressed three-to four-fold by P, and accumulation of the capsid protein and intracellular core particles was similarly suppressed because the pregenomic RNA encodes the capsid protein. Therefore, suppression of the pregenomic RNA by DHBV P creates a negative feedback loop to limit viral antigen accumulation and replication, possibly contributing to maintenance of chronic infection.

Animals↗
PubMed2005

A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame.

BACKGROUND: Hepatitis C virus (HCV) is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription-polymerase chain reaction (RT-PCR) of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile. RESULTS: RT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF) from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon) for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two sets of sequencing primers were optimized for each genotype 1a and 1b. Viral consensus sequences were determined by directly sequencing the amplicons. HCV ORFs from 72 patients have been sequenced using these primers. Sequencing errors were negligible because sequencing depth was over 4-fold and both strands were sequenced. Primer bias was controlled and monitored through careful primer design and control experiments. CONCLUSION: Optimized RT-PCR and sequencing conditions are useful for rapid and reliable amplification and sequencing of HCV genotype 1a and 1b ORFs.

DNA, Complementary↗
PubMed2005

Immune evasion versus recovery after acute hepatitis C virus infection from a shared source.

Acute infection with hepatitis C virus (HCV) rarely is identified, and hence, the determinants of spontaneous resolution versus chronicity remain incompletely understood. In particular, because of the retrospective nature and unknown source of infection in most human studies, direct evidence for emergence of escape mutations in immunodominant major histocompatibility complex class I-restricted epitopes leading to immune evasion is extremely limited. In two patients infected accidentally with an identical HCV strain but who developed divergent outcomes, the total lack of HCV-specific CD4+ T cells in conjunction with vigorous CD8+ T cells that targeted a single epitope in one patient was associated with mutational escape and viral persistence. Statistical evidence for positive Darwinian selective pressure against an immunodominant epitope is presented. Wild-type cytotoxic T lymphocytes persisted even after the cognate antigen was no longer present.

Acute Disease↗
PubMed2005

Identification of an essential molecular contact point on the duck hepatitis B virus reverse transcriptase.

The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.

Amino Acid Sequence↗
PubMed2004

Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization.

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

Animals↗
PubMed2004

Detection and characterization of cytoplasmic hepatitis B virus reverse transcriptase.

It was recently found that the Duck hepatitis B virus (DHBV) reverse transcriptase is primarily a non-encapsidated cytoplasmic molecule that is rapidly translated and has a very short half-life. Here, a non-encapsidated reverse transcriptase from the human Hepatitis B virus (HBV) was characterized. HBV polymerase accumulated in the cytoplasm in a manner similar to non-encapsidated DHBV polymerase. However, the HBV polymerase accumulated at an apparently lower concentration and had a longer half-life than the DHBV enzyme, and it displayed no evidence of the post-translational modifications observed for DHBV. Unlike the DHBV polymerase, immunofluorescence detection of the HBV polymerase in cells was suppressed by the core protein, and this suppression occurred independently of encapsidation. This implies an interaction between the polymerase and core in addition to encapsidation, but the polymerase and core did not co-immunoprecipitate, so the interaction might not be direct. These data indicate that production of cytoplasmic, non-encapsidated polymerase is conserved among the hepadnaviral genera. Furthermore, conservation of the cytoplasmic form of the polymerase suggests that it might have function(s) in virus replication or pathology beyond copying the viral genome.

Cell Line, Tumor↗
PubMed2004

Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting.

The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.

DNA-Directed RNA Polymerases↗
PubMed2003

The duck hepatitis B virus polymerase and core proteins accumulate in different patterns from their common mRNA.

Hepadnaviral reverse transcription occurs in capsids in which the core (C) protein surrounds the reverse transcriptase (P) and pregenomic RNA (pgRNA). We analyzed the accumulation patterns of duck hepatitis B virus P, C, and pgRNA in transfected LMH cells, infected primary duck hepatocytes (PDH), and infected duck liver. In all three systems, P accumulated over time in a different pattern compared with C, despite translation of both proteins from the pgRNA. Although the accumulation patterns of the proteins varied between the systems, in each case P became detectable at the same time or earlier than C and the ratio of P relative to C dropped with time. These accumulation patterns were consistent with the translation rates and half-lives of P and C. Comparing the translation rates of P and C with the pgRNA level over time revealed that translation of P and C was negatively regulated in LMH cells. These data provide a framework for comparing replication studies performed in LMH cells, PDHs and ducks.

Animals↗
PubMed2002

Kinetics of synthesis and turnover of the duck hepatitis B virus reverse transcriptase.

Hepadnaviral reverse transcription occurs in subviral capsids in which the core protein surrounds the reverse transcriptase ("polymerase") and the pregenomic RNA. The pregenomic RNA is the template for reverse transcription and also the bicistronic mRNA for core and polymerase. The pregenomic RNA structure and the capsid stoichiometry imply that vastly more core would be translated than polymerase. Previously, we found that duck hepatitis B virus polymerase unexpectedly accumulates in the cytoplasm (Yao, E., Gong, Y., Chen, N., and Tavis, J. E. (2000) J. Virol. 74, 8648-8657). The production mechanism and function of the excess polymerase are unknown. Here, we determined the kinetics of expression and degradation of polymerase and core in cells producing virus. Polymerase was translated 10% as rapidly as core, the half-life of nonencapsidated polymerase was very short, core had a very long half-life, and very few polymerase molecules were encapsidated. The presence of excess polymerase indicates that the translation rate of the polymerase is not limiting for encapsidation. Therefore, encapsidation must be regulated by other events, most likely binding of the polymerase to the pregenomic RNA. These data support the hypothesis that polymerase may have functions beyond copying the viral genome by demonstrating that the polymerase is a cytoplasmic protein that is only rarely encapsidated.

Half-Life↗
PubMed2000

Evidence that the first strand-transfer reaction of duck hepatitis B virus reverse transcription requires the polymerase and that strand transfer is not needed for the switch of the polymerase to the elongation mode of DNA synthesis.

Deletion of amino acids 79-88 in the duck hepatitis B virus reverse transcriptase had minimal effects on polymerase activities prior to the minus-strand DNA transfer reaction, yet it greatly diminished strand transfer and subsequent DNA synthesis. This mutation also reduced reverse transcription on exogenous RNA templates. The reaction on exogenous RNAs employed the phosphonoformic acid (PFA)-sensitive elongation mode of DNA synthesis rather than the PFA-resistant priming mode, despite the independence of DNA synthesis in this assay from the priming and minus-strand transfer reactions. These data provide experimental evidence that the polymerase is involved directly in the minus-strand transfer reaction and that the switch of the polymerase from the early PFA-resistant mode of DNA synthesis to the later PFA-sensitive elongation mode does not require the strand-transfer reaction.

DNA, Viral↗