MicroRNAs and long non-coding RNAs during transcriptional regulation and latency of HIV and HTLV.

Human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV) have replicative and latent stages of infection. The status of the viruses is dependent on the cells that harbour them and on different events that change the transcriptional and post-transcriptional events. Non-coding (nc)RNAs are key factors in the regulation of retrovirus replication cycles. Notably, micro (mi)RNAs and long non-coding (lnc)RNAs are important regulators that can induce switches between active transcription-replication and latency of retroviruses and have important impacts on their pathogenesis. Here, we review the functions of miRNAs and lncRNAs in the context of HIV and HTLV. We describe how specific miRNAs and lncRNAs are involved in the regulation of the viruses' transcription, post-transcriptional regulation and latency. We further discuss treatment strategies using ncRNAs for HIV and HTLV long remission, reactivation or possible cure.

[Gene therapy to cure HIV infection]. / Thérapie génique pour guérir l'infection par le VIH.

To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

Gene therapy to cure HIV infection.

To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

The interferon-induced protein kinase R: the base of a riboprotein scaffolding regulating the human immunodeficiency virus.

The interferon-induced RNA-activated Protein Kinase (PKR) targets the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α) whose phosphorylation blocks translation initiation of cellular and viral mRNAs. PKR is activated at the beginning of human immunodeficiency virus (HIV) infection by low levels of the HIV Transactivation Response (TAR) RNA and by the cellular PKR Activator (PACT), which contributes to a reduced viral replication. During HIV replication, the viral Tat protein and high production of TAR RNA decrease PKR activation. The cellular TAR RNA Binding Protein (TRBP) and Adenosine Deaminase Acting on RNA (ADAR1) also prevent PKR activation, while HIV expression changes PACT function to become a PKR inhibitor. Therefore, HIV recruits viral and cellular factors to counteract PKR antiviral activity. In addition, PKR antiviral function was positively selected during evolution due to contacts with viral factors inhibiting its function. The riboprotein scaffolding such as the one that inhibits PKR during HIV replication may exist for other antiviral factors.

The interferon-induced protein kinase R: the base of a riboprotein scaffolding regulating the human immunodeficiency virus.

The interferon-induced RNA-activated Protein Kinase (PKR) targets the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α) whose phosphorylation blocks translation initiation of cellular and viral mRNAs. PKR is activated at the beginning of human immunodeficiency virus (HIV) infection by low levels of the HIV Transactivation Response (TAR) RNA and by the cellular PKR Activator (PACT), which contributes to a reduced viral replication. During HIV replication, the viral Tat protein and high production of TAR RNA decrease PKR activation. The cellular TAR RNA Binding Protein (TRBP) and Adenosine Deaminase Acting on RNA (ADAR1) also prevent PKR activation, while HIV expression changes PACT function to become a PKR inhibitor. Therefore, HIV recruits viral and cellular factors to counteract PKR antiviral activity. In addition, PKR antiviral function was positively selected during evolution due to contacts with viral factors inhibiting its function. The riboprotein scaffolding such as the one that inhibits PKR during HIV replication may exist for other antiviral factors.

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

HIV and Ribozymes.

Ribozymes are structured RNA molecules that act as catalysts in different biological reactions. From simple genome cleaving activities in satellite RNAs to more complex functions in cellular protein synthesis and gene regulation, ribozymes play important roles in all forms of life. Several naturally existing ribozymes have been modified for use as therapeutics in different conditions, with HIV-1 infection being one of the most studied. This chapter summarizes data from different preclinical and clinical studies conducted to evaluate the potential of ribozymes to be used in HIV-1 therapies. The different ribozyme motifs that have been modified, as well as their target sites and expression strategies, are described. RNA conjugations used to enhance the antiviral effect of ribozymes are also presented and the results from clinical trials conducted to date are summarized. Studies on anti-HIV-1 ribozymes have provided valuable information on the optimal expression strategies and clinical protocols for RNA gene therapy and remain competitive candidates for future therapy.

The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication.

BACKGROUND: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. RESULTS: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. CONCLUSION: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.

A role for human Dicer in pre-RISC loading of siRNAs.

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.

The discovery and development of RNA-based therapies for treatment of HIV-1 infection.

INTRODUCTION: Long-term control of HIV-1 infection can potentially be achieved using autologous stem cell transplants with gene-modified cells. Non-coding RNAs represent a diverse class of therapeutic agents including ribozymes, RNA aptamers and decoys, small interfering RNAs, short hairpin RNAs, and U1 interference RNAs that can be designed to inhibit HIV-1 replication. They have been engineered for delivery as drugs to complement current HIV-1 therapies and as gene therapies for a potential HIV-1 functional cure. AREAS COVERED: This review surveys the past three decades of development of these RNA technologies with a focus on their efficacy and safety for treating HIV-1 infections. We describe the mechanisms of each RNA-based agent, targets they have been developed against, efforts to enhance their stability and efficacy, and we evaluate their performance in past and ongoing preclinical and clinical trials. EXPERT OPINION: RNA-based technologies are among the top candidates for gene therapies where they can be stably expressed for long-term suppression of HIV-1. Advances in both gene and drug delivery strategies and improvements to non-coding RNA stability and antiviral properties will cooperatively drive forward progress in improving drug therapy and engineering HIV-1 resistant cells.

Enhancement of replication of RNA viruses by ADAR1 via RNA editing and inhibition of RNA-activated protein kinase.

Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA binding protein and RNA-editing enzyme that modifies cellular and viral RNAs, including coding and noncoding RNAs. This interferon (IFN)-induced protein was expected to have an antiviral role, but recent studies have demonstrated that it promotes the replication of many RNA viruses. The data from these experiments show that ADAR1 directly enhances replication of hepatitis delta virus, human immunodeficiency virus type 1, vesicular stomatitis virus, and measles virus. The proviral activity of ADAR1 occurs through two mechanisms: RNA editing and inhibition of RNA-activated protein kinase (PKR). While these pathways have been found independently, the two mechanisms can act in concert to increase viral replication and contribute to viral pathogenesis. This novel type of proviral regulation by an IFN-induced protein, combined with some antiviral effects of hyperediting, sheds new light on the importance of ADAR1 during viral infection and transforms our overall understanding of the innate immune response.