5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.
5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.
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DOI：
10.1128/jvi.01723-21
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发表时间：
2022-03-09
影响因子：
5.4
通讯作者：
Tong S
中科院分区：
文献类型：
--
作者：
Zhang J;Yuan Q;Wang Y;Wang Y;Yuan W;Xia N;Wen Y;Li J;Tong S
B型肝炎病毒（HBV）大（L）包膜蛋白由2.4-kb RNA翻译而来。它含有preS 1、preS 2和S结构域，在Western印迹中检测为p39和gp 42。3.5kb的前基因组RNA产生核心蛋白和聚合酶（P）蛋白。我们从1.1-mer或1.3-mer构建体中产生了基因型A克隆和基因型D克隆的L-减突变体，前者过度产生前基因组RNA。令人惊讶的是，突变preS 1 ATG密码子或引入无义突变之后不久将分泌的p39/gp 42转换为p41/p44双联体，其量通过核心基因中的无义突变进一步增加。一个更下游的preS 1无义突变阻止了p41/p44的产生。衣霉素处理证实p44为p41的糖基化形式。在这方面，基因型D的3.5-kb RNA拼接以在核苷酸（nt）2447至2902处产生连接，能够翻译p43，其中P蛋白的N末端47个残基与L蛋白的C末端371个残基融合。事实上，p41/p44可以通过针对P蛋白N端的抗体检测到，并通过5′ P基因的无义突变或阻止剪接的点突变消除。因此，1.1-mer或1.3-mer构建体中L（和核心）蛋白表达的缺失显著增加了P-L融合蛋白p41/p44（p43）。与L/M蛋白的表达构建体共转染逆转了与L-突变体相关的高细胞外p41/p44，表明L蛋白保留了野生型HBV中的p43，以促进其细胞内降解。考虑到p43缺乏对受体结合至关重要的N端preS 1序列，其在自然感染过程中的生理意义和治疗潜力值得进一步研究。重要性B型肝炎病毒（HBV）的大（L）包膜蛋白由2.4 kb RNA翻译而来，在蛋白质印迹法中检测为p39和gp 42。聚合酶（P）蛋白从3.5-kb RNA以低水平表达。3.5kb RNA的主要剪接形式将在P蛋白的前47个残基和一个短的不相关序列之间产生融合蛋白，尽管也是在低水平上。另一种剪接形式具有相同的P蛋白序列，融合到缺失其前18个残基的L蛋白。我们发现，一些点突变，以消除L和核心蛋白表达从超长HBV DNA构建转化p39/gp 42到p41/gp 44，这原来是P-L融合蛋白。因此，当阻止L蛋白表达时，P-L融合蛋白可以以极高的水平表达。这种L蛋白变体的潜在机制和功能意义值得进一步研究。
Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5′ P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.
DOI：
10.3390/v12090967
发表时间：
2020-09-01
期刊：
Viruses
影响因子：
--
作者：
Wang Q;Fu S;Zhang J;Yuan Q;Li J;Xia N;Wen YM;Wang Y;Tong S
通讯作者：
Tong S