Evaluation of the functional involvement of human immunodeficiency virus type 1 cDNA during integrase in nuclear import of viral acute infection
Evaluation of the functional involvement of human immunodeficiency virus type 1 cDNA during integrase in nuclear import of viral acute infection
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DOI：
10.1128/jvi.78.21.11563-11573.2004
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发表时间：
2004-11-01
影响因子：
5.4
通讯作者：
Masuda, T
中科院分区：
文献类型：
--
作者：
Ikeda, T;Nishitsuji, H;Masuda, T
病毒的核导入是建立人类免疫缺陷病毒1型(HIV-1)前病毒状态的关键步骤。HIV-1整合酶(IN)在病毒DNA核导入中的作用存在争议，部分原因是缺乏对其真实核定位信号的鉴定。在这项研究中，为了解决HIV-1IN的这一假定功能，在病毒复制的背景下，评估了病毒识别的关键残基(PYNP第142至145位、K156、K159和K160)的突变的影响。在急性感染期间，一些突变(N144Q、PYNP>；KL和KKK>；AAA)严重地将病毒基因的表达减少到不到野生型(WT)水平的1%。这些突变均不影响病毒DNA的合成。同时，N144Q、PYNP>；KL和KKK>；aaa突变体产生的整合病毒DNA水平严重下降到wt水平的1%以下。病毒核酸定量聚合酶链式反应和荧光原位杂交分析表明，这些突变显著降低了病毒DNA在细胞核内的积聚水平。进一步的分析表明，携带N144Q、PYNP>；KL和KKK>；AAA突变的IN蛋白与病毒的结合严重减少，但保持了它们的亲核特性。综上所述，这些结果表明，降低IN与病毒cDNA结合的突变导致病毒感染性的严重损害，很可能是通过影响进行整合的病毒cDNA的核输入。这些结果表明，HIV-1IN可能是有效的病毒DNA核导入的关键成分之一。
Nuclear import of viral cDNA is a critical step for establishing the proviral state of human immunodeficiency virus type 1 (HIV-1). The contribution of HIV-1 integrase (IN) to the nuclear import of viral cDNA is controversial, partly due to a lack of identification of its bona fide nuclear localization signal. In this study, to address this putative function of HIV-1 IN, the effects of mutations at key residues for viral cDNA recognition (PYNP at positions 142 to 145, K156, K159, and K160) were evaluated in the context of viral replication. During acute infection, some mutations (N144Q, PYNP > KL, and KKK > AAA) severely reduced viral gene expression to less than 1% the wild-type (WT) level. None of the mutations affected the synthesis of viral cDNA. Meanwhile, the levels of integrated viral cDNA produced by N144Q, PYNP > KL, and KKK > AAA mutants were severely reduced to less than 1% the WT level. Quantitative PCR analysis of viral cDNA in nuclei and fluorescence in situ hybridization analysis showed that these mutations significantly reduced the level of viral cDNA accumulation in nuclei. Further analysis revealed that IN proteins carrying the N144Q, PYNP > KL, and KKK > AAA mutations showed severely reduced binding to viral cDNA but kept their karyophilic properties. Taken together, these results indicate that mutations that reduced the binding of IN to viral cDNA resulted in severe impairment of virus infectivity, most likely by affecting the nuclear import of viral cDNA that proceeds integration. These results suggest that HIV-1 IN may be one of the critical constituents for the efficient nuclear import of viral cDNA.