Mutagenic primers.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

N-连接糖基化（N-linked glycosylation）是一种关键的翻译后修饰，可显著影响蛋白质生物学的诸多方面。细胞自噬（Autophagy）作为一类重要的细胞过程，在细胞存活与维持中发挥核心作用。乙型肝炎病毒（hepatitis B virus, HBV）已演化出调控该过程的机制，以确保其在宿主细胞内的存活。尤为重要的是，HBV大表面蛋白（large surface protein of HBV, LHBs）上的翻译后N-连接糖基化，可对病毒粒子组装、感染性以及免疫逃逸产生影响。本研究探讨了LHBs的N-连接糖基化在细胞自噬中的作用，及其对HBV复制与分泌的后续影响。研究人员通过在N4、N112及N309位点进行氨基酸替换，构建了携带单、双、三重N-连接糖基化位点突变的LHBs质粒。与野生型LHBs相比，包括N309Q、N4-309Q、N112-309Q及N4-112-309Q在内的N-糖基化突变体，可诱导肝癌细胞中的自噬基因表达，并引发自噬体积累。对表达LHBs突变体的细胞进行吖啶橙染色后发现，溶酶体酸化过程受损，这提示自噬流在后期阶段可能被阻断。此外，N-糖基化突变体可上调HBV表面抗原（HBV surface antigen, HBsAg）的mRNA表达水平。值得注意的是，N309Q可显著提升HBx癌基因的表达水平。LHBs突变体（尤其是N309Q与N112-309Q）可显著增强HBV复制，而N309Q、N4-309Q及N4-112-309Q则可显著增加HBV子代病毒的分泌量。值得一提的是，通过3-甲基腺嘌呤（3-methyladenine, 3-MA）抑制剂实验证实，细胞自噬对于LHBs的N-连接糖基化突变影响HBV分泌的过程不可或缺。本研究为解析LHBs的N-连接糖基化突变、宿主细胞自噬与HBV生命周期之间的相互作用提供了开创性见解。此外，本研究还为进一步探究肝细胞癌（hepatocellular carcinoma, HCC）的致癌机制提供了新的思路。上述研究结果凸显了靶向N-连接糖基化修饰或自噬通路，以开发针对HBV及/或肝细胞癌的创新疗法的潜在价值。