Department of Molecular Biology & Biochemistry, University of California, Irvine, California, USA Division of Infectious Disease, University of California, Irvine, California, USA Cancer Research Institute, University of California, Irvine, California, USA Center for Virus Research, University of California, Irvine, California, USA Institute for Immunology, University of California, Irvine, California, USA.
Division of Infectious Disease, University of California, Irvine, California, USA Cancer Research Institute, University of California, Irvine, California, USA Center for Virus Research, University of California, Irvine, California, USA Institute for Immunology, University of California, Irvine, California, USA.
J Virol. 2014 Jun;88(12):7054-69. doi: 10.1128/JVI.00704-14. Epub 2014 Mar 26.
Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different factors and occur in different intracellular locations.
This study shows that HIV-1 reverse transcription starts in the cytoplasm but is completed in or on the surface of the nucleus. Moreover, we show that nuclear reverse transcription is dependent on the activity of the human RNA lariat debranchng enzyme (DBR1), while cytoplasmic reverse transcription is not. These findings may provide new avenues for inhibiting HIV-1 replication and therefore may lead to new medicines for treating HIV-1-infected individuals.
先前的研究表明，短发夹 RNA（shRNA）敲低 RNA 套索分支酶（DBR1）会导致 HIV-1 cDNA 的产生减少。为了进一步描述这种效应，将 DBR1 shRNA 引入 GHOST-R5X4 细胞中，然后在接近 HIV-1 或 HIV-1 衍生载体的单位感染倍数下感染。在感染后不同时间从整个细胞以及细胞质和核部分分离 DNA 和 RNA。DBR1 的抑制对负链强终止 cDNA 的形成几乎没有影响，但导致中间和全长 cDNA 的形成显著减少。此外，负链强终止 DNA 在感染后 2 小时内迅速在细胞质中积累，但在感染后 6 小时转移到核部分。无论 DBR1 是否受到抑制，在所有时间点，中间长度和全长 HIV-1 cDNA 都大于 95%存在于核部分。因此，在这些实验条件下，HIV-1 cDNA 合成在细胞质中起始，并在感染细胞的核或核周区域完成。当通过表达 mRNA 切割和多聚腺苷酸化因子 CPSF6 的截断形式阻断 HIV-1 逆转录复合物的核输入时，在细胞质中检测到 HIV-1 载体 cDNA 合成的完成，并且它不受 DBR1 敲低的抑制。对核分离程序的改进表明，逆转录的完成既发生在核内，也发生在核周区域。总之，结果表明，在接近 1 的多重感染中，HIV-1 逆转录在感染细胞的核或核周区域完成，这依赖于 DBR1。当核转运被抑制时，逆转录以 DBR1 不依赖的方式在细胞质中完成。因此，HIV-1 逆转录至少有两种机制，需要不同的因素，并发生在不同的细胞内位置。
本研究表明，HIV-1 逆转录从细胞质开始，但在核内或核表面完成。此外，我们表明，核逆转录依赖于人 RNA 套索分支酶（DBR1）的活性，而细胞质逆转录则不然。这些发现可能为抑制 HIV-1 复制提供新的途径，从而可能为治疗 HIV-1 感染个体提供新的药物。
