Molecular, Evolutionary, and Structural Analysis of the Terminal Protein Domain of Hepatitis B Virus Polymerase, a Potential Drug Target
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viruses Review Molecular, Evolutionary, and Structural Analysis of the Terminal Protein Domain of Hepatitis B Virus Polymerase, a Potential Drug Target Timothy S. Buhlig 1 , Anastasia F. Bowersox 2, Daniel L. Braun 2, Desiree N. Owsley 1, Kortney D. James 1 , Alfredo J. Aranda 1, Connor D. Kendrick 1, Nicole A. Skalka 1 and Daniel N. Clark 1,* 1 Microbiology Department, Weber State University, 1415 Edvalson St., Ogden, UT 84408, USA; [email protected] (T.S.B.); [email protected] (D.N.O.); [email protected] (K.D.J.); [email protected] (A.J.A.); [email protected] (C.D.K.); [email protected] (N.A.S.) 2 Biology Department, Lebanon Valley College, 101 N. College Ave., Annville, PA 17003, USA; [email protected] (A.F.B.); [email protected] (D.L.B.) * Correspondence: [email protected]; Tel.: +1-801-626-6948 Received: 15 April 2020; Accepted: 19 May 2020; Published: 22 May 2020 Abstract: Approximately 250 million people are living with chronic hepatitis B virus (HBV) infections, which claim nearly a million lives annually. The target of all current HBV drug therapies (except interferon) is the viral polymerase; specifically, the reverse transcriptase domain. Although no high-resolution structure exists for the HBV polymerase, several recent advances have helped to map its functions to specific domains. The terminal protein (TP) domain, unique to hepadnaviruses such as HBV, has been implicated in the binding and packaging of the viral RNA, as well as the initial priming of and downstream synthesis of viral DNA—all of which make the TP domain an attractive novel drug target. This review encompasses three types of analysis: sequence conservation analysis, secondary structure prediction, and the results from mutational studies. It is concluded that the TP domain of HBV polymerase is comprised of seven subdomains (three unstructured loops and four helical regions) and that all three loop subdomains and Helix 5 are the major determinants of HBV function within the TP domain. Further studies, such as modeling inhibitors of these critical TP subdomains, will advance the TP domain of HBV polymerase as a therapeutic drug target in the progression towards a cure. Keywords: hepatitis B virus; terminal protein; protein priming 1. Introduction Hepatitis B virus (HBV) infects the liver and is commonly spread through sexual, blood, and vertical contact. It is a leading cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. Although several hundred million people in the world are infected by HBV, an effective vaccine exists, as well as non-curative treatment [1]. Current antiviral drugs, except immunomodulatory drugs such as pegylated interferon α and thymalfasin, are all directed against the HBV polymerase (Pol) [2–4], though therapeutics for novel targets are in development [5]. Although there is no small animal model capable of infection with human HBV, informative studies have been performed using related HBV species that infect avian and mammalian hosts, such as duck HBV (DHBV) and woodchuck HBV [6]. With the discovery of the receptor for HBV (human sodium taurocholate co-transporting polypeptide receptor (hNTCP or SLC10A1)) [7], the ability to create transgenics in small animals and cell culture systems is also leading to advances in model systems [8]. Viruses 2020, 12, 570; doi:10.3390/v12050570 www.mdpi.com/journal/viruses Viruses 2020, 12, x 2 of 19 Viruses 2020, 12, 570 2 of 19 transporting polypeptide receptor (hNTCP or SLC10A1)) [7], the ability to create transgenics in small animals and cell culture systems is also leading to advances in model systems [8]. HBVHBV contains contains a a smallsmall circular circular DNA DNA genome, genome, reverse reverse transcribed transcribed by the by HBV the Pol HBV from Pol viral from RNA viral RNA[9]. [The9]. TheHBV HBV Pol protein Pol protein is a major is a major focus focusof basic of basicand translational and translational research; research; indeed, indeed, it is the itonly is the onlycurrent current target target of ofany any HBV-specific HBV-specific antiviral antiviral and and the the only only region region commonly commonly sequenced sequenced during during treatmenttreatment escape escape [ 10[10].]. AlthoughAlthough the the catalytic catalytic activi activityty of of HBV HBV Pol Pol occurs occurs within within the RT the and RT andRNase RNase H Hdomains (Figure (Figure 11),), the TP domain also shows cons considerableiderable functional functional utility. utility. The The TP TP domain domain is is uniqueunique to toHepadnaviridae Hepadnaviridae, and, and therefore, therefore, anyany drugdrug cross-reactivitycross-reactivity would would likely likely be be low. low. FigureFigure 1. 1.Schematic Schematic representation of of hepatitis hepatitis B virus B virus polymerase polymerase domains domains and terminal and terminal protein protein (TP) (TP)subdomains. subdomains. The The polymerase polymerase protein protein is responsible is responsible for DNA for DNA synthesis, synthesis, which which is carried is carried out by outthe by thecatalytic catalytic reverse reverse transcriptase transcriptase (RT) (RT) domain. domain. While While the RNA the RNA template template is copied is copied into viral into DNA, viral DNA,the theRNase RNase H H domain domain degrades degrades the the RNA RNA template. template. The The TP TP domain domain acts acts as asa primer a primer for for the the initial initial DNA DNA synthesissynthesis steps, steps, and and the the DNA DNA remains remains attachedattached toto the TP domain throughout throughout synthesis. synthesis. The The spacer spacer domaindomain is is thought thought to to allow allow for for flexibility flexibility whilewhile the TP is attached attached at at one one end end of of the the nascent nascent viral viral DNA DNA andand the the RT domainRT domain synthesizes synthesizes the other the end.other Regionsend. Re ofgions similarity of similarity with other with proteins other areproteins highlighted. are Withinhighlighted. the TP Within domain the (green), TP domain the function(green), the of thefunction helical of the subdomains helical subdomains is likely tois likely provide to provide structure, whilestructure, the loop while subdomains the loop aresubdomains involved are in activities involvedcritical in activities to the critical viral replication to the viral cycle, replication such as cycle, protein primingsuch as that protein initiates priming from that the tyrosineinitiates atfrom position the tyro 63sine (Y63). at Theposition regions 63 (Y63). of the The TP domainregions thatof the overlap TP withdomain other that open overlap reading with frames other (ORF) open reading are shown frames as striped (ORF) are boxes. shown These as striped regions boxes. are under Theseadditional regions selectionare under pressure additional when selection mutations pressure occur. when HBV: mutations hepatitis B occur. virus; HBV: HIV: humanhepatitis immunodeficiency B virus; HIV: human virus; Pol:immunodeficiency polymerase. virus; Pol: polymerase. DownstreamDownstream of of the the TP TP domain domain areare thethe spacer, reverse transcriptase transcriptase (RT), (RT), and and RNase RNase H Hdomains domains (Figure(Figure1). 1). HBV HBV Pol Pol is is similar similar in in sequence sequence andand structurestructure to the polymerase found found in in the the human human immunodeficiency virus (HIV,) which also contains RT and RNase H domains. Indeed, emtricitabine, immunodeficiency virus (HIV,) which also contains RT and RNase H domains. Indeed, emtricitabine, lamivudine, and tenofovir are able to inhibit both HBV and HIV [9]. Much more is known of the HIV lamivudine, and tenofovir are able to inhibit both HBV and HIV [9]. Much more is known of the Pol, including several high-resolution structures. Due to their similarity, models of HBV Pol borrow HIV Pol, including several high-resolution structures. Due to their similarity, models of HBV Pol this knowledge about the HIV Pol [11,12]. Despite similarities, definitive descriptions of the three- borrow this knowledge about the HIV Pol [11,12]. Despite similarities, definitive descriptions of the dimensional structure of HBV Pol do not yet exist. Additionally, HBV’s TP and spacer domains are three-dimensionalunique to the Hepadnaviridae structure of HBV family. Pol Currently, do not yet their exist. structures Additionally, can only HBV’s be predicted. TP and spacer domains are uniqueTP proteins to the Hepadnaviridae also exist in other family. microbes; Currently, ho theirwever, structures they give can clue onlys to be predicted.the function—not necessarilyTP proteins the alsostructure—of exist in other HBV microbes; Pol. For however,example, bacteriophage, they give clues members to the function—not of the Adenoviridae necessarily thefamily, structure—of and Streptomyces HBV Pol. bacteria For are example, groups bacteriophage,of microbes that membersencode a TP of protein the Adenoviridae for priming DNA family, andsynthesisStreptomyces [13–15].bacteria Typical are among groups these of microbes TP proteins that encode is the ause TP of protein a tyrosine, for priming serine, DNA or threonine synthesis for [13 – 15].initiating Typical priming among [16,17]. these TP Beyond proteins these is thepriming use of residues, a tyrosine, little serine, amino or acid threonine homology for initiatingcan be primingidentified [16, 17among]. Beyond TP proteins these priming (Figure 1). residues, One commo little aminonality