TAR loop-dependent human immunodeficiency virus trans activation requires factors encoded on human chromosome 12.

The trans-activator response region (TAR) RNA in the human immunodeficiency virus type 1 (HIV-1) and HIV-2 long terminal repeat forms stem-loop secondary structures in which the loop sequence is essential for trans activation. We investigated how the HIV trans-activation mechanism encoded on human chromosome 12 relates to the TAR RNA loop-dependent pathway. DNA transfection experiments showed that trans activation in human-hamster hybrid cells with the single human chromosome 12 and human T-cell lines was highly dependent on the native sequences of the HIV-1 TAR loop and the HIV-2 5' TAR loop. In nonhuman cell lines or hybrid cells without chromosome 12 that supported trans activation, the cellular mechanism was independent of the HIV-1 TAR loop and the response to mutations in the HIV-2 TAR loops differed from that found in human T-cell lines and human-hamster hybrid cells with chromosome 12. Our results suggest that the human chromosome 12 mechanism interacts directly with the TAR RNA loop or indirectly by regulating TAR RNA-binding proteins.     

反式激活应答元件RNA在HIV-1感染中的作用

反式激活应答(transactivation response,TAR)元件RNA作为HIV-1中的一种非编码RNA,从转录与翻译水平负调控HIV-1的基因表达.同时HIV-1采取了相应的策略拮抗TAR RNA的负调控作用:病毒蛋白Tat或细胞蛋白TAR RNA结合蛋白(TRBP)结合TAR RNA后,分别在转录与翻译水平促进HIV-1的基因表达.此外,TAR编码的miRNA有助于保持HIV的潜伏感染及阻止细胞凋亡.TAR与其它蛋白间相互作用及其功能的研究对于深入了解HIV-1感染细胞后的调控机制,寻求新的抗HIV治疗靶点具有重要意义.     

Lentiviral-mediated delivery of combined HIV-1 decoy TAR and Vif siRNA as a single RNA molecule that cleaves to inhibit HIV-1 in transduced cells

RNA interference (RNAi) silences gene expression via short interfering 21-23 mer double-stranded RNA (siRNA) segments that guide cognate mRNA degradation in a sequence-specific manner. On the other hand, HIV-1 decoy TAR RNA are known to competitively interact with the HIV-1 Tat protein, to downregulate the enhanced gene expression from the long terminal repeat (LTR) promoters. Here we report that a novel expression construct, encoding both HIV-1 decoy TAR and Vif siRNA, as a single RNA substrate, was expressed under the control of the human U6 promoter, and later the TAR and siRNA were cleaved into their respective separate RNA by the endogenous RNase III-like enzyme. Each of the cleaved HIV-1 anti-genes then synergistically contributed toward enhancing the inhibition efficacy (>80%) of HIV-1 replication in transduced Jurkat cells. These results suggest that targeting HIV-1 mRNA with simultaneously expressed intracellular decoy TAR and Vif-siRNA could lead to an effective gene therapy strategy for the control and management of HIV-AIDS.     

LENTIVIRAL-MEDIATED DELIVERY OF COMBINED HIV-1 DECOY TAR AND Vif siRNA AS A SINGLE 1RNA MOLECULE THAT CLEAVES TO INHIBIT HIV-1 IN TRANSDUCED CELLS

RNA interference (RNA_i) silences gene expression via short interfering 21–23 mer double-stranded RNA (siRNA) segments that guide cognate mRNA degradation in a sequence-specific manner. On the other hand, HIV-1 decoy TAR RNA are known to competitively interact with the HIV-1 Tat protein, to downregulate the enhanced gene expression from the long terminal repeat (LTR) promoters. Here we report that a novel expression construct, encoding both HIV-1 decoy TAR and Vif siRNA, as a single RNA substrate, was expressed under the control of the human U6 promoter, and later the TAR and siRNA were cleaved into their respective separate RNA by the endogenous RNase III-like enzyme. Each of the cleaved HIV-1 anti-genes then synergistically contributed toward enhancing the inhibition efficacy (> 80%) of HIV-1 replication in transduced Jurkat cells. These results suggest that targeting HIV-1 mRNA with simultaneously expressed intracellular decoy TAR and Vif-siRNA could lead to an effective gene therapy strategy for the control and management of HIV-AIDS.     

The basic RNA-binding domain of HIV-2 Tat contributes to preferential trans-activation of a TAR2-containing LTR.

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that trans-activates the respective viral genome through RNA targets (TAR1 and TAR2). Tat-1 and Tat-2 have considerable homology. However, an interesting biological observation has been that Tat-1 activates the HIV-1 and HIV-2 LTRs equally while Tat-2 activates the former, in comparison to the latter, poorly. Here, we present evidence that it is the TAR2 RNA target together with the basic RNA-binding protein domain of Tat-2 that dictate this non-reciprocity in trans-activation.     

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.     

Kinetics of HIV-1 long terminal repeat trans-activation. Use of intragenic ribozyme to assess rate-limiting steps.

We have examined, using self-cleaving ribozymes, the intracellular trans-activation kinetics of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by viral protein Tat. Experiments were designed to effect a competition (during RNA chain elongation) between cleavage of a nascent RNA containing the Tat-responsive target sequence (TAR) and Tat interaction with the same TAR in the process of LTR-trans-activation. We found that fast self-cleavage of nascent TAR-containing RNA abolished Tat trans-activation. Slowing the cleavage reaction kinetically rescued trans-activation. Based on our results, we conclude that the rate-limiting step in HIV-1 LTR trans-activation is the initial contact made between Tat/TAR/LTR rather than the promoter proximal pausing of RNA polymerases that are tethered to functional TAR.