Human cells infected with HIV Genome Sequencing

TNPO3, a karyopherin specific for serine/arginine (SR)-rich splicing factors, increases the efficiency of transduction by HIV-1 and most lentiviruses, but not by MLV. Chimeric vectors generated by exchanging HIV-1 and MLV components and functional assessment of HIV-1 capsid mutant viruses showed capsid to be the viral determinant of TNPO3-dependence. CPSF6-358 is a truncated SR-protein lacking the putative nuclear localization signal that inhibits HIV-1 replication in a capsid-dependent manner. 27 HIV-1 capsid substitution mutants were tested for sensitivity to inhibition by CPSF6-358. The effect of the capsid mutants on sensitivity to inhibition by CPSF6-358 correlates with sensitivity to TNPO3 knockdown of the same panel of viruses. To determine if HIV-1 inhibition by TNPO3 knockdown (KD) results from CPSF6 accumulation in the cytoplasm, CPSF6-358 and full-length CPSF6 were expressed as fusions with the SV40 large T antigen nuclear localization signal (NLS) or with the protein kinase inhibitor nuclear export signal (NES). CPSF6-358-NLS did not inhibit HIV-1 transduction whereas CPSF6-NES did. When CPSF6 was depleted, TNPO3 KD no longer inhibited HIV-1 transduction. Rescue of the CPSF6 KD with non-targetable CPSF6-NLS did not restore the inhibitory effect of TNPO3 KD. Biochemical experiments indicated that the accumulation of CPSF6 in the cytoplasm, whether by TNPO3 KD or by the CPSF6-358 truncation, resulted in abnormally stable capsids. Our results demonstrate that the effect of TNPO3 KD on HIV-1 replication results from effects on the subcellular localization of capsid-binding protein CPSF6.

TNPO3是一种特异性结合富含丝氨酸/精氨酸（SR）剪接因子的核转运蛋白（karyopherin），可提升人类免疫缺陷病毒1型（HIV-1）及多数慢病毒的转导效率，但对鼠白血病病毒（MLV，Murine Leukemia Virus）无此作用。通过交换HIV-1与MLV组分构建的嵌合载体，以及对HIV-1衣壳突变病毒的功能验证实验均证实，衣壳是决定病毒对TNPO3依赖性的病毒源性因子。CPSF6-358是一种截短型SR蛋白，缺失了推定的核定位信号，可通过衣壳依赖的方式抑制HIV-1的复制。本研究针对27株HIV-1衣壳替换突变株，检测其对CPSF6-358抑制作用的敏感性。这些衣壳突变株对CPSF6-358抑制作用的敏感性差异，与同一组病毒对TNPO3敲低（KD）的敏感性呈显著相关。为探究TNPO3敲低对HIV-1的抑制作用是否源于CPSF6在细胞质中的蓄积，本研究将CPSF6-358与全长CPSF6分别与猿猴空泡病毒40（SV40）大T抗原核定位信号（NLS）或蛋白激酶抑制剂核输出信号（NES）构建为融合蛋白进行表达。结果显示，CPSF6-358-NLS无法抑制HIV-1的转导，而CPSF6-NES则可产生抑制效果。当CPSF6被敲除后，TNPO3敲低便不再对HIV-1的转导产生抑制作用。使用无法被靶向敲低的CPSF6-NLS回补CPSF6敲低模型，未能恢复TNPO3敲低的抑制效应。生化实验证实，无论是通过TNPO3敲低还是CPSF6-358截短导致的CPSF6细胞质蓄积，均会使病毒衣壳呈现异常稳定的状态。本研究结果证实，TNPO3敲低对HIV-1复制产生的影响，源于其对衣壳结合蛋白CPSF6的亚细胞定位的调控作用。