Rescue of Multiple Viral Functions by a Second-Site Suppressor of a Human Immunodeficiency Virus Type 1 Nucleocapsid Mutation

Human immunodeficiency type 1 (HIV-1) bearing the nucleocapsid (NC) mutation R10A/K11A is replication defective. After serial passage of the mutant virus in tissue culture, we isolated a revertant that retained the original mutation. It had acquired, in addition, a new mutation (E21K) that was formally demonstrated to be sufficient for restoration of viral replication. Detailed analysis of the replication defect of R10A/K11A revealed a threefold reduction in virion yield and a fivefold reduction in packaging of viral genomic RNA. Real-time PCR was then used to quantitate viral DNA synthesis following infection of Jurkat T cells. After adjustment for the assembly and packaging defects, a minor (twofold) reduction in synthesis of either strong-stop, full-length linear DNA or 2-LTR circles was observed with R10A/K11A virions, indicating that reverse transcription and nuclear transport of the viral genome were largely intact. However, after adjustment for the amounts of full-length or 2-LTR circles produced, R10A/K11A virions were at least 10-fold less infectious than wild type, indicating that viral DNA produced by the R10A/K11A mutant failed to integrate. Each of the above-mentioned defects was corrected by introduction of the second-site compensatory mutation E21K. These results demonstrate that the replication defect of mutant R10A/K11A can be explained by impairment at multiple steps in the viral life cycle, most important among them being integration and RNA packaging. The E21K mutation is predicted to restore positive charge to the face of the R10A/K11A mutant NC protein that interacts with the HIV-1 SL3 RNA stem-loop, emphasizing the importance of NC basic residues for HIV-1 replication.

携带核衣壳（NC）R10A/K11A突变的人类免疫缺陷病毒1型（HIV-1）存在复制缺陷。我们将该突变病毒在组织培养中进行连续传代后，分离得到一株保留了原始突变的回复突变株；该回复突变株额外获得了E21K新突变，经正式证实该突变足以恢复病毒的复制能力。
对R10A/K11A突变株的复制缺陷开展详细分析后发现，其病毒粒子产量下降3倍，病毒基因组RNA的包装效率降低5倍。随后我们采用实时PCR（Real-time PCR）定量检测感染Jurkat T细胞后的病毒DNA合成水平。在校正组装与包装缺陷后，R10A/K11A病毒粒子的强终止全长线性DNA以及2-LTR环的合成仅出现小幅（2倍）下降，提示该突变株的逆转录过程及病毒基因组的核转运基本完好。
然而，在对所产生的全长DNA或2-LTR环的量进行校正后，R10A/K11A病毒粒子的感染性至少较野生型低10倍，表明R10A/K11A突变株产生的病毒DNA无法完成整合过程。
上述所有缺陷均可通过引入第二位点补偿突变E21K得到修复。本研究结果显示，R10A/K11A突变株的复制缺陷可归因于病毒生命周期多个步骤的功能受损，其中最为关键的缺陷为整合过程与RNA包装。E21K突变可恢复R10A/K11A突变株NC蛋白的正电荷表面特性，该蛋白区域负责与HIV-1 SL3 RNA茎环结构（SL3 RNA stem-loop）相互作用，这一结果凸显了NC碱性残基对于HIV-1复制的重要性。