Galvis Alvaro E, Fisher Hugh E, Fan Hung, Camerini David
Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA.
Division of Infectious Diseases, University of California, Irvine, California, USA.
J Virol. 2017 Nov 14;91(23). doi: 10.1128/JVI.01377-17. Print 2017 Dec 1.
Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it. Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.
我们实验室之前的研究表明，RNA去分支酶（DBR1）在HIV cDNA形成的早期步骤中并非必需，但对于中间和晚期cDNA产物的合成是必需的。为了进一步表征这种效应，我们评估了在早期感染期间，有无抑制DBR1合成时HIV-1 RNA基因组5'端的拓扑结构。用DBR1短发夹RNA（shRNA）转染细胞，48小时后用含有RNase H缺陷逆转录酶（RT）的HIV-1衍生载体感染。在感染后的几个时间点分离RNA，并在对HIV-1 RNA的5' cDNA末端进行快速扩增（5' RACE）和定量逆转录酶PCR（qRT-PCR）之前，用各种RNA修饰酶处理。在感染的细胞中，DBR1敲低在所有时间点均抑制了游离HIV-1 RNA 5'端的检测。在5' RACE和qRT-PCR之前，将感染细胞的RNA与酵母或人DBR1酶孵育，消除了感染的DBR1敲低细胞与对照细胞中游离HIV-1 RNA 5'端检测的差异。这取决于DBR1的2'-5'磷酸酶活性，因为当我们使用催化无活性的DBR1（N85A）突变体时不会发生这种情况。最后，来自感染的DBR1敲低细胞的HIV-1 RNA对降解线性RNA但不降解环状或套索状构象RNA的RNase R具有抗性。这些结果为感染早期涉及HIV-1 RNA 5'端的套索状结构的形成以及DBR1参与解决该结构提供了证据。我们的发现支持了HIV基因组复制早期步骤的新观点。我们表明，HIV基因组RNA进入细胞后会迅速脱帽并形成套索状结构。随后，套索状结构被细胞酶DBR1分解，留下一个5'磷酸基团。该途径类似于前体mRNA内含子套索的形成和分解，因此表明HIV可能使用类似的机制。因此，我们的工作开辟了HIV复制研究的新领域，并最终可能揭示抑制HIV复制和预防艾滋病发展的新靶点。
