Protein Priming Research Articles

Unveiling the roles of HBV polymerase for new antiviral strategies.

Infection with HBV is common worldwide, with over 350 million chronic carriers. Chronic HBV infection is associated with cirrhosis and hepatocellular carcinoma. All currently available oral antivirals are directed against the HBV polymerase enzyme, a reverse transcriptase. HBV polymerase contains several important domains and motifs which define its functions and reveal ways to further target it. This enzyme executes many functions required for the HBV replication cycle, including viral RNA binding, RNA packaging, protein priming, template switching, DNA synthesis and RNA degradation. In addition, HBV polymerase must interact with host proteins for its functions. Future therapeutics may inhibit not only the DNA synthesis steps which are carried out by the reverse transcriptase domain (as all current antivirals do) but other domains, functions and interactions which are essential to the HBV replication cycle.

Monocyte-derived dendritic cells promote T follicular helper cell differentiation.

To be effective, protein priming must induce the development of a distinct lineage of CD4+ T cells named T follicular helper (Tfh) cells, which regulate the differentiation of high-affinity memory B cells and long-lived plasma cells. In this context, we tested how adjuvantation with CpG, the Toll-like receptor 9 agonist used in clinics, contributes to antigen-specific T-cell-dependent B-cell responses in vivo. We found that addition of CpG to other vaccine adjuvant increased the differentiation of antigen-specific Tfh cells without changing the overall magnitude of the T-cell response. This phenomenon correlated with an enhancement of the germinal centre reaction, antigen-specific plasma cells and circulating antibodies. We comprehensively demonstrated that, in addition to the classical Tfh-cell differentiation mediated by conventional DC, the promoting effect due to CpG was orchestrated in vivo by antigen presentation and IL-6 secreted by monocyte-derived dendritic cells (DC) as shown in their absence. Thus, while conventional DC initiate T-cell responses, targeting monocyte-derived DC specifically enhances the Tfh programme needed to regulate high-affinity B-cell protection in vivo.

Structural basis for the replication of genomic RNA of porcine enteric sapovirus by VPg (LB243)

Structural basis for the replication of genomic RNA of porcine enteric sapovirus by VPg Hye Jung Min, Hyo‐Jeong Hwang, Jeong‐Sun Kim, and Chul Won Lee Department of Chemistry, Chonnam National University, Gwangju 500‐757, Korea. Viral protein genome‐linked (VPg) plays a critical role in the life cycle of vertebrate and plant positive‐sense (+) RNA viruses by acting as a protein primer for RNA synthesis. The replication of the viruses is primed by the VPg protein by attaching to the 5’‐end of the viral RNA genome. Here we show the solution structure of VPg of porcine enteric sapovirus (PSV) determined by multi‐dimensional NMR spectroscopy. The structure of VPg comprises three helices (denoted α1, α2, and α3) which were tightly packed together through hydrophobic interactions formed by several hydrophobic residues to form a three‐helix bundle structure. The orientation and length of three helices of PSV VPg are similar to those of feline calicivirus (FCV) VPg with similar core structure formed by conserved hydrophobic residues. The tyrosine residue (Tyr956) within the conserved motif for nucleotide acceptor is exposed to the solvent surface to facilitate nucleotidylation by viral RNA polymerases. These results may provide the molecular basis for the replication of positive‐RNA viruses.

Comparative Analysis of Hepatitis B Virus Polymerase Sequences Required for Viral RNA Binding, RNA Packaging, and Protein Priming

Hepatitis B virus replicates a DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific association with a viral RNA signal, termed ε (Hε), located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. HP is made up of four domains, including the terminal protein (TP), the spacer, the reverse transcriptase (RT), and the RNase H domains. A recently developed, Hε-dependent, in vitro protein priming assay was used in this study to demonstrate that almost the entire TP and RT domains and most of the RNase H domain were required for protein priming. Specific residues within TP, RT, and the spacer were identified as being critical for HP-Hε binding and/or protein priming. Comparison of HP sequence requirements for Hε binding, pgRNA packaging, and protein priming allowed the classification of the HP mutants into five groups, each with distinct effects on these complex and related processes. Detailed characterization of HP requirements for these related and essential functions of HP will further elucidate the mechanisms of its multiple functions and aid in the targeting of these functions for antiviral therapy.

Noncompetitive Inhibition of Hepatitis B Virus Reverse Transcriptase Protein Priming and DNA Synthesis by the Nucleoside Analog Clevudine

Noncompetitive Inhibition of Hepatitis B Virus Reverse Transcriptase Protein Priming and DNA Synthesis by the Nucleoside Analog Clevudine

Noncompetitive Inhibition of Hepatitis B Virus Reverse Transcriptase Protein Priming and DNA Synthesis by the Nucleoside Analog Clevudine

All currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.

Uniplex and duplex PCR detection of geminivirus associated with potato apical leaf curl disease in India

Apical leaf curl disease has emerged as a new disease in potato during the last decade in India due to a change in planting date and an increased whitefly population. Its incidence is on the rise threatening the cultivation of potato across the country. Hence, a PCR assay was developed for the detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV-Potato) which is the causal agent of apical leaf curl disease in potato. Primers specific to the coat protein (AV1) and replicase (AC1) gene regions were designed and used for standardization of the PCR. Some of the primers (LCVCPF1/LCVCPR1, LCVREPF2/LCVREPR2, LCrep1F/LCrep2R) could detect the virus in 2.4–0.24pg of total DNA of infected plant. A duplex PCR assay was optimized with the selected coat protein gene specific primers and primers specific to potato urease gene, a housekeeping gene served as an internal check. The suitability of these primers was examined for detection of the virus in 80 potato apical leaf curl disease samples from 11 different potato growing states of India and also from micro-plants grown in tissue culture. The selected coat protein primer pair (LCVCPF1/LCVCPR1) was found to be conserved in all 80 isolates except for a few isolates, which had a single nucleotide substitution in the forward primer sequence. These substitutions did not interfere with amplification of the coat protein gene. The primers could detect the virus using a print-capture PCR assay both in the presence and absence of an internal control. These results indicate the robustness of the PCR assay for virus indexing of mother stocks in the seed production system.

The Streptococcus thermophilus protein Wzh functions as a phosphotyrosine phosphatase

Amino acid residues that are important for metal binding and catalysis in Gram-positive phosphotyrosine phosphatases were identified in the Wzh protein of Streptococcus thermophilus MR-1C by using sequence comparisons. A His-tagged fusion Wzh protein was purified from Escherichia coli cultures and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine-threonine peptides. Purified Wzh released 2316.5 ± 138.7 pmol PO4·min(-1)·μg(-1) from phosphotyrosine peptide-1 and 2345.7 ± 135.2 pmol PO4·min(-1)·μg(-1) from phosphotyrosine peptide-2. The presence of the phosphotyrosine phosphatase inhibitor sodium vanadate decreased purified Wzh activity by 45%-50% at 1 mmol·L(-1), 74%-84% at 5 mmol·L(-1), and by at least 88% at 10 mmol·L(-1). Purified Wzh had no detectable activity against the phosphoserine-threonine peptide. These results clearly establish that S. thermophilus MR-1C Wzh functions as a phosphotyrosine phosphatase that could function to remove phosphate groups from proteins involved in exopolysaccharide biosynthesis, including the protein tyrosine kinase Wze and priming glycosyltransferase.

Polyinosinic-Polycytidylic Acid Is the Most Effective TLR Adjuvant for SIV Gag Protein–Induced T Cell Responses In Nonhuman Primates

Prime-boost immunization with heterologous vaccines elicits potent cellular immunity. In this study, we assessed the influence of various TLR ligands on SIV Gag-specific T cell immunity and protection following prime-boost immunization. Rhesus macaques (RMs) were primed with SIV Gag protein emulsified in Montanide ISA51 with or without TLR3 (polyinosinic-polycytidylic acid [poly-IC]), TLR4 (monophosphoryl lipid A), TLR7/8 (3M-012), TLR9 (CpG), or TLR3 (poly-IC) combined with TLR7/8 ligands, then boosted with replication defective adenovirus 5 expressing SIV Gag (rAd5-Gag). After priming, RMs that received SIV Gag protein plus poly-IC developed significantly higher frequencies of SIV Gag-specific CD4(+) Th1 responses in blood and bronchoalveolar lavage (BAL) fluid lymphocytes compared with all other adjuvants, and low-level SIV Gag-specific CD8(+) T cell responses. After the rAd5-Gag boost, the magnitude and breadth of SIV Gag-specific CD8(+) T cell responses were significantly increased in RM primed with SIV Gag protein plus poly-IC, with or without the TLR7/8 ligand, or CpG. However, the anamnestic, SIV Gag-specific CD8(+) T cell response to SIVmac251 challenge was not significantly enhanced by SIV Gag protein priming with any of the adjuvants. In contrast, the anamnestic SIV Gag-specific CD4(+) T cell response in BAL was enhanced by SIV Gag protein priming with poly-IC or CpG, which correlated with partial control of early viral replication after SIVmac251 challenge. These results demonstrate that prime-boost vaccination with SIV Gag protein/poly-IC improves magnitude, breadth, and durability of CD4(+) T cell immune responses, which could have a role in the control of SIV viral replication.

Tests for the mechanism of starch biosynthesis: de novo synthesis or an amylogenin primer synthesis

Studies in 1940 on potato phosphorylase reaction with starch found that d-glucopyranose from α-d-glucopyranosyl-1-phosphate was added to the nonreducing-ends of starch chains. This led to the hypothesis that the biosynthesis of starch required a preformed primer. Later it was found that phosphorylase was exclusively a degradative enzyme in vivo and that starch-synthase was the enzyme that reacted with ADPGlc to biosynthesize starch. Amylogenin, a putative self-glycosylated protein, was postulated to be the primer, although it was never demonstrated or found. In the present study, three reactions were performed in sequence with a highly purified potato starch-synthase to determine whether an amylogenin primer was present and required or whether the biosynthesis was de novo. Reaction 1 was performed by adding 2.0mM ADPGlc to synthesize the putative primer to a possible amylogenin in the preparation; in Reaction 2, 10mM ADP-[14C]Glc was added; and in Reaction 3, 10mM nonlabeled ADPGlc was added. After the isolation, reduction, and acid hydrolysis of the products of Reactions 2 and 3, 14C-d-glucitol was obtained from Reaction 2 and was decreased by Reaction 3. The formation of 14C-d-glucitol and its decrease showed that an amylogenin, protein primer was not involved in starch biosynthesis and the synthesis is de novo by the addition of d-glucose to the reducing-ends of growing starch chains.

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

Hepatitis B virus (HBV) replication requires reverse transcription of an RNA pregenome (pgRNA) by a multifunctional polymerase (HP). HP initiates viral DNA synthesis by using itself as a protein primer and an RNA signal on pgRNA, termed epsilon (Hε), as the obligatory template. We discovered a Mn(2+)-dependent transferase activity of HP in vitro that was independent of Hε but also used HP as a protein primer. This protein-primed transferase activity was completely dependent on the HP polymerase active site. The DNA products of the transferase reaction were linked to HP via a phosphotyrosyl bond, and replacement of the Y63 residue of HP, the priming site for templated DNA synthesis, almost completely eliminated DNA synthesis by the transferase activity, suggesting that Y63 also serves as the predominant priming site for the transferase reaction. For this transferase activity, HP could use all four deoxynucleotide substrates, but TTP was clearly favored for extensive polymerization. The transferase activity was highly distributive, leading to the synthesis of DNA homo- and hetero-oligomeric and -polymeric ladders ranging from 1 nucleotide (nt) to >100 nt in length, with single-nt increments. As with Hε-templated DNA synthesis, the protein-primed transferase reaction was characterized by an initial stage that was resistant to the pyrophosphate analog phosphonoformic acid (PFA) followed by PFA-sensitive DNA synthesis, suggestive of an HP conformational change upon the synthesis of a nascent DNA oligomer. These findings have important implications for HBV replication, pathogenesis, and therapy.

Rapid development of cross-clade neutralizing antibody responses after clade B gp120/gp140 protein priming and clade c gp140 protein boosting

Background Immunization with heterologous Env protein immunogens following an immunologic rest period has the potential to generate cross-clade neutralizing antibody responses. We identified individuals who had received a clade B Env protein with MF59 4-17 years earlier, most in combination with a DNA or ALVAC prime, and administered a clade C protein boost in an open label phase 1 trial. Methods

Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 μM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

First report of association of a begomovirus with the leaf curl disease of a common weed, datura inoxia.

Begomovirus is one of the largest genus of the family Geminiviridae and the vector white fly (Bemisia tabaci) is prevalent in the tropical and subtropical regions of the world [4]. Weeds may infect crops and those weeds may serve as a reservoir for crop-infecting geminiviruses [1, 5]. In the present report we identified a begomovirus infecting Datura inoxia, a common hallucinogenic alkaloid containing toxic weed found in India annually. D. inoxia showing typical begomovirus symptoms i.e., leaf curling, decrease in leaf size, and growth stunting as well as healthy leaves were collected from the fields of Rajasthan, India (Fig. 1). The DNA was extracted from infected leaves using CTAB method and PCR was carried out by using universal coat protein primer AC 1048 and AV 494 [2]. A typical PCR contained about 2.5 μl DNA template, 2.5 μl Taq 10× buffer (10 mM Tris–HCl, pH 8.8, 50 mM KCl and 1 % [v/v]), 1.5 μl MgCl2, 2 μl of each dNTPs, 0.5 μl of Taq DNA Polymerase, and 1 μl of each primer. The PCR thermal profile were pre-PCR denaturation at 94 °C for 60 s followed by 35 cycles of denaturing at 94 °C for 60 s, annealing at 55 °C for 2 min and extension at 72 °C for 3 min, and a final extension at 72 °C for 5 min. The PCR product was partially sequenced ({type:entrez-nucleotide,attrs:{text:JN000702,term_id:334847691,term_text:JN000702}}JN000702) and 550 nucleotides sequence was used to construct a phylogenetic tree using the neighbor-joining method in DNASIS Pro ver. 2.6. In order to confirm the geminivirus infectivity, Southern blotting was performed according to the method of Kon et al. [3]. Cloned DNA-A (2.7 Kb) of Chilli leaf curl virus was used to develop the radio-labeled probes. Fig. 1 Datura inoxia showing leaf curl symptoms in Lakshmangarh, Rajasthan in 2011 Sequence analysis of the virus under study showed 82–83 % nucleotide sequence identity with the corresponding region of Chilli leaf curl virus ({type:entrez-nucleotide,attrs:{text:FM210476,term_id:199601437,term_text:FM210476}}FM210476), Tomato leaf curl virus ({type:entrez-nucleotide,attrs:{text:AJ810360,term_id:62815995,term_text:AJ810360}}AJ810360), and Croton yellow vein virus ({type:entrez-nucleotide,attrs:{text:FN543112,term_id:304939561,term_text:FN543112}}FN543112). In neighbor-joining tree, the coat protein gene of the virus in datura leaf curl formed a separate monophyletic cluster with a low bootstrap value (Fig. 2). To complement the information the amplified product was used for the Southern hybridization. The strong signal showed that the virus in D. inoxia had some homology with Chilli leaf curl virus. The result suggested that the virus in D. inoxia samples belonged to the genus begomovirus. To the best of our knowledge, this is the first report of begomovirus associated with leaf curl of D. inoxia. Fig. 2 Neighbor-Joining tree based on the partial sequence of coat protein gene ({type:entrez-nucleotide,attrs:{text:JN000702,term_id:334847691,term_text:JN000702}}JN000702), of the virus isolate obtained from leaf curl disease of Datura inoxia ...

TP-RT Domain Interactions of Duck Hepatitis B Virus Reverse Transcriptase in cis and in trans during Protein-Primed Initiation of DNA Synthesis In Vitro

The hepadnavirus reverse transcriptase (RT) has the unique ability to initiate viral DNA synthesis using RT itself as a protein primer. Protein priming requires complex interactions between the N-terminal TP (terminal protein) domain, where the primer (a specific Y residue) resides, and the central RT domain, which harbors the polymerase active site. While it normally utilizes the cis-linked TP to prime DNA synthesis (cis-priming), we found that the duck hepatitis B virus (DHBV) RT domain, in the context of the full-length RT protein or a mini-RT construct containing only truncated TP and RT domains, could additionally use a separate TP or RT domain in trans as a primer (trans-priming). trans interaction could also be demonstrated by the inhibitory effect (trans-inhibition) on cis-priming by TP and RT domain sequences provided in trans. Protein priming was further shown to induce RT conformational changes that resulted in TP-RT domain dissociation, altered priming site selection, and a gain of sensitivity to a pyrophosphate analog inhibitor. trans-priming, trans-inhibition, and trans-complementation, which requires separate TP and RT domains to reconstitute a functional RT protein, were employed to define the sequences in the TP and RT domains that could mediate physical or functional inter- and intradomain interactions. These results provide new insights into TP-RT domain interactions and conformational dynamics during protein priming and suggest novel means to inhibit protein priming by targeting these interactions and the associated conformational transitions.

In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.

Distinct families of cis-acting RNA replication elements epsilon from hepatitis B viruses

The hepadnavirus encapsidation signal, epsilon (ε), is an RNA structure located at the 5′ end of the viral pregenomic RNA. It is essential for viral replication and functions in polymerase protein binding and priming. This structure could also have potential regulatory roles in controlling the expression of viral replicative proteins. In addition to its structure, the primary sequence of this RNA element has crucial functional roles in the viral lifecycle. Although the ε elements in hepadnaviruses share common critical functions, there are some significant differences in mammalian and avian hepadnaviruses, which include both sequence and structural variations. Here we present several covariance models for ε elements from the Hepadnaviridae. The model building included experimentally determined data from previous studies using chemical probing and NMR analysis. These models have sufficient similarity to comprise a clan. The clan has in common a highly conserved overall structure consisting of a lower-stem, bulge, upper-stem and apical-loop. The models differ in functionally critical regions—notably the two types of avian ε elements have a tetra-loop (UGUU) including a non-canonical UU base pair, while the hepatitis B virus (HBV) epsilon has a tri-loop (UGU). The avian epsilon elements have a less stable dynamic structure in the upper stem. Comparisons between these models and all other Rfam models, and searches of genomes, showed these structures are specific to the Hepadnaviridae. Two family models and the clan are available from the Rfam database.

Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage ϕ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of ϕ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized.

Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.