Virion Secretion of Hepatitis B Virus Naturally Occurring Core Antigen Variants
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cells Review Virion Secretion of Hepatitis B Virus Naturally Occurring Core Antigen Variants Chiaho Shih 1,2,*, Szu-Yao Wu 3, Shu-Fan Chou 4 and Ta-Tung Thomas Yuan 5,* 1 Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan 2 Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan 3 Chimera Bioscience Inc., No. 18 Siyuan St., Zhongzheng Dist., Taipei 10087, Taiwan; [email protected] 4 Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA; [email protected] 5 TFBS Bioscience, Inc. 3F, No. 103, Ln 169, Kangning St., Xizhi Dist., New Taipei City 221, Taiwan * Correspondence: [email protected] (C.S.); [email protected] (T.-T.T.Y.) Abstract: In natural infection, hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations. The most frequent HBc variant in chronic hepatitis B patients is mutant 97L, changing from an isoleucine or phenylalanine to a leucine (L) at HBc amino acid 97. One dogma in the HBV research field is that wild type HBV secretes predominantly virions containing mature double- stranded DNA genomes. Immature genomes, containing single-stranded RNA or DNA, do not get efficiently secreted until reaching genome maturity. Interestingly, HBc variant 97L does not follow this dogma in virion secretion. Instead, it exhibits an immature secretion phenotype, which preferentially secretes virions containing immature genomes. Other aberrant behaviors in virion secretion were also observed in different naturally occurring HBc variants. A hydrophobic pocket around amino acid 97 was identified by bioinformatics, genetic analysis, and cryo-EM. We postulated that this hydrophobic pocket could mediate the transduction of the genome maturation signal for envelopment from the capsid interior to its surface. Virion morphogenesis must involve interactions between HBc, envelope proteins (HBsAg) and host factors, such as components of ESCRT (endosomal sorting complex required for transport). Immature secretion can be offset by compensatory mutations, Citation: Shih, C.; Wu, S.-Y.; Chou, occurring at other positions in HBc or HBsAg. Recently, we demonstrated in mice that the persistence S.-F.; Yuan, T.T. Virion Secretion of of intrahepatic HBV DNA is related to virion secretion regulated by HBV genome maturity. HBV Hepatitis B Virus Naturally virion secretion could be an antiviral drug target. Occurring Core Antigen Variants. Cells 2021, 10, 43. https://doi.org/ Keywords: hepatitis B virus (HBV); naturally occurring mutation; HBV core antigen; immature 10.3390/cells10010043 virion secretion; genome maturation; hydrophobic pocket; compensatory mutation; persistence; hydrodynamic mouse model Received: 8 November 2020 Accepted: 28 December 2020 Published: 30 December 2020 Publisher’s Note: MDPI stays neu- 1. Introduction tral with regard to jurisdictional clai- Hepatitis B virus (HBV) is a major human pathogen [1,2]. Chronic infection with ms in published maps and institutio- HBV could lead to chronic hepatitis, cirrhosis and hepatoma [3,4]. At present, no curative nal affiliations. treatment can effectively eradicate the virus from patients [5–7]. HBV replicates via a sloppy polymerase containing a reverse transcriptase domain with low fidelity in DNA synthesis. Therefore, HBV in chronic carriers exists as a quasispecies, rather than one single homogeneous population. The most frequent naturally occurring mutation in HBV core Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. protein (HBc) occurs at amino acid 97, changing an isoleucine or phenylalanine to leucine This article is an open access article (I97L or F97L). This hotspot mutation was found in HBV DNA in the sera of Japanese distributed under the terms and con- hepatitis B patients with liver injury, or severe fulminant hepatitis [8–10], as well as in the ditions of the Creative Commons At- integrated HBV DNA in Taiwanese hepatoma samples (Figure1)[ 11,12]. In asymptomatic tribution (CC BY) license (https:// carriers and self-limited acute hepatitis B, this mutation was not found. Furthermore, this creativecommons.org/licenses/by/ HBc 97L mutation is present not only in Asian patients but also in Italians with chronic 4.0/). Cells 2021, 10, 43. https://doi.org/10.3390/cells10010043 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 20 Cells 2021, 10, 43 tomatic carriers and self-limited acute hepatitis B, this mutation was not found. Further-2 of 19 more, this HBc 97L mutation is present not only in Asian patients but also in Italians with chronic infection [13]. Therefore, 97L mutation can be found in different ethnic groups in different areas of the world. infection [13]. Therefore, 97L mutation can be found in different ethnic groups in different areas of the world. Figure 1. Coincidence of naturally occurring HBc mutations with mapped T cell epitopes in literature. Large dots, more Figurethan 45%; 1. Coincidence medium dots, of 20%naturally to 35%; occu andrring small HBc dots, mutations fewer than with 20% mapped of HBV-infected T cell epitopes patients in literature. are found toLarge have dots, virus more with thana predominant 45%; medium mutation dots, 20% at this to position35%; and (adapted small dots, with fewer permission than 20% from of Hosono HBV-infected et al., 1995) patients [12]. are found to have virus with a predominant mutation at this position (adapted with permission from Hosono et al., 1995) [12]. What could be the functional significance of this HBc mutation at amino acid 97? HosonoWhat et could al. [12 ]be reported the functional that HBc significance mutations of at codonsthis HBc 5, mutation 97 and 130, at allamino coincide acid with97? HosonoHLA class et al. II-restricted [12] reported T cell that epitopes. HBc mutations In the woodchuck at codons model5, 97 and infected 130, all with coincide woodchuck with HLAhepatitis class virus II-restricted (WHV), T peptides cell epitopes. 1–20, In 97–110, the woodchuck and 112–131 model were infected shown with to be woodchuck major T-cell hepatitisepitopes virus of woodchuck (WHV), peptides hepatitis 1–20, B core 97–110, antigen and 112–131 [14]. Similarly, were shown peptide to be 91–105, major whichT-cell epitopesoverlaps of with woodchuck amino acid hepatitis 97, produced B core antigen maximal [14]. proliferation Similarly, ofpeptide the peripheral 91–105, which blood overlapslymphocytes with fromamino chronic acid 97, WHV produced carriers maxima [15]. Althoughl proliferation naturally of the occurring peripheral HBc blood mutation lym- phocytescould fall from within chronic the target WHV epitopes carriers of [15]. CTL Although (cytotoxic naturally T lymphocytes) occurring in human HBc mutation patients, couldmore fall direct within and the rigorous target evidenceepitopes of for CTL this (cytotoxic immune escapeT lymphocytes) interpretation in human remains patients, to be morefurther direct examined and rigorous in animal evidence models. for this immune escape interpretation remains to be further examined in animal models. 2. An Immature Secretion Phenotype of Mutant 97L 2. An BeyondImmature its Secretion potential significancePhenotype of in Mutant immunity, 97L mutant 97L displays pleiotropic phe- notypes.Beyond In its particular, potential onesignificance dogma in immunity, the field is mutant that, for 97L a displays wild type pleiotropic hepadnavirus, phe- notypes.only virions In particular, containing one a maturedogma genomein the field (Figure is that,2A) for are a preferentially wild type hepadnavirus, secreted into only the medium [16]. This dogma is violated by mutant 97L. For wild type HBV in cell culture, the virions containing a mature genome (Figure 2A) are preferentially secreted into the me- ratio between the secreted mature genome and immature genome is generally around 4 dium [16]. This dogma is violated by mutant 97L. For wild type HBV in cell culture, the to 1. For this variant 97L, it secretes almost equal amounts (1:1) of mature and immature ratio between the secreted mature genome and immature genome is generally around 4 genomes. Therefore, one major characteristic feature of mutant 97L is its secretion of to 1. For this variant 97L, it secretes almost equal amounts (1:1) of mature and immature excessive amount of virions containing immature genomes (Figure2B). In other words, genomes. Therefore, one major characteristic feature of mutant 97L is its secretion of ex- mutant I97L lost the high stringency of selectivity in genome maturity during virion export. cessive amount of virions containing immature genomes (Figure 2B). In other words, mu- This is a widely observed phenotype since it is true in HBV subtypes ayw [17], adr [18] tant I97L lost the high stringency of selectivity in genome maturity during virion export. and genotype A [19]. In addition, immature secretion is an evolutionarily conserved phe- This is a widely observed phenotype since it is true in HBV subtypes ayw [17], adr [18] and nomenon found in other non-human hepadnaviruses, such as snow goose hepatitis B virus genotype A [19]. In addition, immature secretion is an evolutionarily conserved phenom- (SGHBV) [20,21]. In a woodchuck hepatitis B virus (WHV) model, when WHV-infected enon found in other non-human hepadnaviruses, such as snow goose hepatitis B virus woodchucks were treated with acyclovir, minus-strand viral DNA were enveloped and (SGHBV)secreted as[20,21]. virion-like In a woodchuck particles [22 hepatitis]. B virus (WHV) model, when WHV-infected woodchucks were treated with acyclovir, minus-strand viral DNA were enveloped and secreted as virion-like particles [22]. Cells 2021, 10, x FORCells PEER2021 REVIEW, 10, 43 3 of 20 3 of 19 Figure 2. HBV core variants 97L exhibited abnormal behaviors in viral DNA synthesis and virion secretion. (A) Cartoon Figure 2. HBV core variants 97L exhibited abnormal behaviors in viral DNA synthesis and virion illustrations of mature and immature HBV genomes. pgRNA: pregenomic RNA; SS DNA: single-strand (−) DNA reversed secretion. (A) Cartoon illustrations of mature and immature HBV genomes. pgRNA: pregenomic transcribed from pgRNA (+); RC DNA: partially double-strand relaxed circle DNA.