Explore hepatitis B virus preS2 mutants as immune escape mutants

摘要 慢性乙肝病毒感染是导致肝癌的主要原因。乙肝病毒产生 共末端的大(L)、中(M)、小(S)被膜蛋白，含有PreS1+PreS2+S、PreS2+S、 和S单独域名。中央S结构域和N端前S1结构域分别介导 病毒粒子附着在低亲和力和高亲和力的乙肝病毒受体HSPG和NTCP上。对应 抗体可以中和乙肝病毒的传染性，尽管抗前S2抗体如何中和乙肝病毒的传染性 仍然是个谜。有趣的是，阻止M蛋白表达或人为删除大部分 前S2结构域不影响细胞培养中乙肝病毒粒子的产生或病毒的感染性，并不影响晚期 慢性乙肝病毒感染常选择前S2 ATG密码子点突变来消除M蛋白 表达，或框内缺失，以移除N-末端的preS2结构域。这样的删除要多得多 在肝癌患者中比轻度肝病患者更常见，但其驱动力尚不清楚。 这项R21探索性拨款申请旨在描述这种天然生物的生物学特性 发生的和临床上重要的乙肝病毒变异。我们假设1)M-减去突变体和preS2 缺失突变体仍然具有感染性；2)前S2缺失逃脱了抗前S2抗体的中和， 它可以通过与L或M蛋白上的前S2结构域结合来发挥作用；3)从而M- 突变体部分逃脱了抗preS2抗体。提出了两个具体目标，以批判性地测试我们的 工作中的假设。目标1将描述临床上重要的M-阴性和前S2缺失突变体 并确定它们的传染性。自然产生的突变将被引入具有感染性的克隆人 C型和D型，以及从转染的HepG2细胞中产生的病毒颗粒 将其接种于HepG2/NTCP细胞和分化后的HepaRG细胞，检测其感染力。目标2 将确定这类M--和preS2缺失突变体对中和抗体和 确定抗前S2抗体是否通过L、M或任何一种蛋白起作用。我们将废除M蛋白 通过突变preS2 ATG密码子进行表达，并通过优化Kozak序列增强其表达。 此外，含有中和的病毒颗粒逃脱了L、M或两种蛋白中的前S2缺失，将 通过共转染实验产生，并测定它们对抗前S2抗体的敏感性。 如果拟议的研究表明，抗前S2抗体可以通过M蛋白中和乙肝病毒的传染性， 那么，另一个宿主因素可能参与了野生型乙肝病毒的感染。在这方面聚合了 人血清白蛋白可以与M蛋白上的preS2结构域结合，这是30多年来提出的 作为一种进入乙肝病毒的机制。

ABSTRACT Chronic infection with hepatitis B virus (HBV) is a leading cause of liver cancer. HBV produces co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1+preS2+S, preS2+S, and S domain alone, respectively. Central S domain and N-terminal preS1 domain respectively mediate virion attachment to HSPG and NTCP, the low- and high-affinity HBV receptors. Corresponding antibodies can neutralize HBV infectivity, although how anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. Intriguingly, preventing M protein expression or artificially deleting most part of the preS2 domain does not impair HBV virion production or viral infectivity in cell culture, and late stage of chronic HBV infection often selects for point mutation of preS2 ATG codon to abolish M protein expression, or in-frame deletions to remove N-terminal preS2 domain. Such deletions are much more prevalent in liver cancer patients than those with mild liver diseases, but the driving force remains unclear. This R21 exploratory grant application aims to characterize the biological properties of such naturally occurring and clinically important HBV mutants. We hypothesize that 1) M-minus mutants and preS2 deletion mutants remain infectious; 2) preS2 deletions escape neutralization by anti-preS2 antibodies, which can work through binding to preS2 domain on either L or M protein; 3) consequently M-minus mutants partially escape anti-preS2 antibodies. Two specific aims are proposed to critically test our working hypotheses. Aim 1 will characterize clinically important M-minus and preS2 deletion mutants and establish their infectivity. The naturally occurring mutations will be introduced to an infectious clone of genotype C and genotype D, respectively, and viral particles generated from transfected HepG2 cells will be inoculated to HepG2/NTCP cells and differentiated HepaRG cells to test their infectivity. Aim 2 will determine susceptibility of such M-minus and preS2 deletion mutants to neutralizing antibodies and establish whether anti-preS2 antibodies work through L, M, or either protein. We will abolish M protein expression by mutation of preS2 ATG codon and enhance its expression via optimized Kozak sequence. In addition, viral particles containing neutralization escaping preS2 deletion in L, M, or both proteins will be generated by co-transfection experiments and their susceptibility to anti-preS2 antibodies determined. If the proposed studies reveal that anti-preS2 antibodies can neutralize HBV infectivity through M protein, then an additional host factor might be involved in infection by wild-type HBV. In this regard polymerized human serum albumin can bind to the preS2 domain on M protein, which was proposed over 30 years ago as an HBV entry mechanism.