Discovery And Significance Study Of A Novel Functional Region In Hepatitis B Virus Polymerase

Discovery and significance study of a novel functional region in hepatitis B virus polymeraseHepatitis B virus (HBV) infects more than 300 million people in the wholeworld. HBV belongs to Hepadnaviridae, which is the family with a partially doublestranded and circular DNA genome. HBV replicates via a unique reversetranscription process. The structure and function of the polymerase/reversetranscriptase (RT) play important roles in HBV replication. Unfortunately, to date,active HBV RT with enzymatic functions can not be produced in sufficient amountfor in vitro biochemical study and crystallographic analysis. Besides, there is yetno in vitro infection system available to analyze the complex replication cycle ofHBV. Through full-length genome cloning and cell transfection based replicationefficiency assay of two genotype B HBV strains isolated from hepatitis B patients,we have previously reported that when proline at residue 306 (rtP306) in HBV RTwas substituted by serine or other amino acids, most of the mutants showeddecreased replicative competency. To explore the mechanisms for the decreaseof replicative competency in these substituted mutants, mutated constructssubstituting rtP306 of a genotype C strain were used to transfect Huh-7 cells, andreplication competency, transcription levels as well as encapsidation efficiencywere compared between the mutants and the parental viral strain. Decreasedreplication efficiency of rtP306 mutant was found by cell transfection basedreplication efficiency assay, which was further confirmed bytrans-complementation study between the RT mutants and an RT-deleted repliconrtW58^*.To further study the mechanisms for the decreased replication competency,the wild-type virus and its mutants were first analyzed for their transcriptionallevels. No changes in transcriptional level were detected in all mutated constructs. Furthermore, substitutions at rtP306 did not affect RT stability by Western blotting.Therefore, two major functions of HBV RT-participating in pgRNA encapsidationand nucleotidyl transfer activity to produce progeny virus DNA were analyzed byspecific pgRNA encapsidation assay and endogenous polymerase reaction (EPR).The pgRNA encapsidation competencies of these constructs were studied bymonitoring the pregenomic RNA (pgRNA) in intracellular core particles fromtransfected cells, which were normalized with the amount of HBV core particles byWestern blotting using anti-core antibodies. Hampered pgRNA encapsidationefficiency was found in most of the substituted mutants. EPR results confirmedthe impaired pgRNA encapsidation efficiency of rtP306 mutants and showed thatin some rtP306 mutants reduced nucleotidyl transfer activity was also involved.Based on the above findings, we speculated that aside from only one amino acidinvolved, other residues flanking rtP306 (rt304-311) might also contribute toefficient HBV replication. By constructing mutants substituting the flankingresidues (rt304-311) of rtP306 with alanine or phenylalanine and assay for theirreplication efficiencies, we found that substitutions at rt304-311 indeed led todrastically decreased replication efficiency of HBV. The mechanisms involvedwere also shown as no changes at the transcriptional level or RT stability, butdecreased pgRNA encapsidation efficiency. In contrast, substitutions at residues(rt336 and rt346) distal from rtP306, no changes in their replication competencieswere detected. To our knowledge, this is the first time to reveal that residueconservation of rt304-311 is crucial for efficient encapsidation of pgRNA. Inaddition, compared to the parental virus, substitution at rt305 (rtY305A) showedreduced sensitivity to one anti-HBV drug-adefovir. As adefovir was reported to acton the functional active site in the predicted "palm" subdomain of RT, whilesubstitution at rtY305 was at the predicted "thumb" subdomain, result suggestedthat substitutions at rt304-311 would alter the conformation of HBV RT resulting indiminished affinity to anti-HBV drugs.Bioinformatic analysis was used for evaluation of the implication of rt304-311in HBV RT and HBV replication. Results showed that 1) residues rt304-311 werepredicted at the turn of an helix-turn-helix (named "helix clamp") motif in the thumb subdomain of HBV RT; 2) the turn residues were considerably conserved amongall reported genotype A-H HBV strains; 3) several turn residues of HBV RT werealso conserved at the helix clamp motif of retroviral RT and RNA dependent RNApolymerase of hepatitis C virus. Helix clamp motif was found as a commonstructure element in the "thumb" domain of nucleic acid polymerases ofeukaryotes, bacteria and viruses and could bind nucleic acid template and primerduring polymerization. Hence, the turn (rt304-311) of helix clamp motif of HBV RTwas both confirmed by biological studies and structural bioinformatic analysis.Thus, this turn of helix clamp motif was discovered as a novel functional region toregulate pgRNA encapsidation efficiency of HBV, which might be mediated bycontrolling the binding affinity of HBV RT to the nucleic acid.Human immunodeficiency virus (HIV) is the prototype of lentivirus genus ofRetroviridae. Similar to HBV, HIV also replicates via a reverse transcriptionprocess. To study whether the turn of helix clamp motif of HIV RT could also playa pivotal role in HIV-1 replication, substitutions of the turn residues of HIV RTrelative to that of HBV RT with alanine were carried out in a HIV-1 provirusplasmid. The replication capacity of the plasmids with substituted alanine wasmonitored by one cycle infection assay. Preliminary results showed that mutantswith substitutions, namely, rtY271A and rtl274A, were not able to replicate inJurkat cells. Furthermore, HIV-1 RT mutants, namely, rtY271A and rtl274A,displayed decreased binding affinity to DNA template/primer. Taken together, thepredicted turn of helix clamp motif of HBV RT as well as that of HIV RT playcrucial roles in viral replication and could be used a new antiviral target.To date, the registered anti-HBV drugs such as lamivudine, adefovir dipivoxil andentecavir are nucleosid(t)e analog inhibitors targeting catalytic centre of HBV RT,The prolonged use of these drugs usually would lead to emergence of drugresistant HBV mutants. Hence, there is the urgent need for developing novelanti-HBV drugs. In an effort to screen the compounds targeting the turn of helixclamp motif of HBV RT or HIV RT, a non-nucleoside reverse transcriptase inhibitor(NNRTI), 2-naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)] azo, (KM-1)), was reported highly potent to inhibit HIV RT with a mechanismdifferent from the known NNRTIs. Furthermore, the targeting site of KM-1 waspredicted at the thumb subdomain of HIV RT. Hence, KM-1 was evaluated bymultiple assays for its anti-HBV activity. By cell-free endogenous polymerasereaction, KM-1 was shown to inhibit not only the wild type HBV RT but also itsmutant rtL180M/M2041, which is resistant to triphosphorylated lamivudine. Therespective 50% inhibitory concentration (IC₅₀) was 2.6μM and 0.5μM, and KM-1was confirmed to block HBV RT with a mechanism different from that oftriphosphorylated lamivudine. However, by cell transfection based replicationassay, KM-1 inhibited the replication efficiencies of wild type HBV and its mutantrtL180M/M2041 with the respective IC₅₀ of 31.2μM and 9.5μM. Besides, KM-1displayed moderate cytotoxicity with a 50% cytotoxic concentration of 105μM. Itis expected that by designing new compounds with high binding efficiencies to thethumb region or even to the turn of RT thumb, new anti-HBV or anti-HIV drugs canbe developed.