Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (ΔIN) or 34 C-terminal amino acid residues (Δ34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Δ34 and ΔIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Δ34 and ΔIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of ΔIN mutant virions could not be complemented with the Δ34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

逆转录病毒DNA的整合酶（integrase, IN）结构域发生突变可能影响病毒生命周期的多个阶段，提示IN蛋白除整合功能外，还可能具备其他生物学功能。我们此前曾报道，人类免疫缺陷病毒1型（human immunodeficiency virus type 1, HIV-1）的IN蛋白对于高效完成病毒DNA合成是必需的，且该功能依赖于与其他病毒组分的特异性相互作用，而非酶（整合）活性。本研究针对莫洛尼鼠白血病病毒（Moloney murine leukemia virus, MLV）的IN蛋白在病毒DNA合成过程中的结构与功能进行了系统解析。我们采用搭载绿色荧光蛋白（green fluorescent protein, GFP）作为病毒感染灵敏报告因子的MLV载体开展实验，结果发现，催化三联体（D184A）或HHCC基序（H61A）发生突变的病毒，其感染性均下降约1000倍。完整缺失IN序列（ΔIN）或缺失C端34个氨基酸残基（Δ34）的突变体则表现出更为严重的缺陷，感染性水平持续降低达10000倍。免疫印迹分析结果表明，相较于野生型MLV，上述突变体在病毒粒子生成以及Gag与Pol前体蛋白的蛋白水解加工环节上均无显著差异。我们采用半定量聚合酶链式反应（semiquantitative PCR）分析感染细胞内的病毒cDNA合成情况，发现Δ34与ΔIN突变体的cDNA合成能力显著受损，而D184A和H61A突变体的cDNA合成水平与野生型病毒基本一致。若以Gag-IN融合蛋白形式在反式环境中补充野生型IN或D184A突变型IN，可挽救Δ34与ΔIN突变体的DNA合成缺陷。但ΔIN突变病毒粒子的DNA合成缺陷无法通过Δ34突变型IN实现反式互补。综合上述实验结果，我们可以明确得出结论：MLV的IN蛋白本身对于高效完成病毒DNA合成是必需的，且该功能可能在其他逆转录病毒中具有进化保守性。