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 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.

Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus.

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction.

Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters.

The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5' end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5' transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.

Cloning and Detection of Aptamer-Ribozyme Conjugations.

RNA aptamers can be used to target proteins or nucleic acids for therapeutic purposes and are candidates for RNA-mediated gene therapy. Like other small therapeutic RNAs, they can be expressed in cells from DNA templates that include a cellular promoter upstream of the RNA coding sequence. Secondary structures flanking aptamers can be used to enhance the activity or stability of these molecules. Notably, flanking self-cleaving ribozymes to remove extraneous nucleotides included during transcription as well as flanking hairpins to improve RNA stability have been used to increase the effect of therapeutic aptamers. Here we describe the cloning procedure of aptamers containing different flanking secondary structures and methods to compare their expression levels by a northern blot protocol optimized for the detection of small RNA molecules.

A U1i RNA that Enhances HIV-1 RNA Splicing with an Elongated Recognition Domain Is an Optimal Candidate for Combination HIV-1 Gene Therapy.

U1 interference (U1i) RNAs can be designed to correct splicing defects and target pathogenic RNA, such as HIV-1 RNA. In this study, we show that U1i RNAs that enhance HIV-1 RNA splicing are more effective at inhibiting HIV-1 production compared to top U1i RNAs that inhibit polyadenylation of HIV-1 RNA. A U1i RNA was also identified targeting a site upstream of the first splice acceptor site in the Gag coding region that was effective at inhibiting HIV-1 production. U1-T6, which enhanced HIV-1 RNA splicing, was superior to an antiviral short hairpin RNA (shRNA) currently in clinical trials. To increase specificity, the recognition domain of U1-T6 was elongated by 3-6 nt. The elongated molecules inhibited HIV-1 production from different HIV-1 strains, including one with a mismatch in the target site. These results suggest that lengthening the recognition domain can enhance the specificity of U1i RNAs for their intended target sites while at the same time allowing them to tolerate single mismatch mutations. Overall, our results demonstrate that U1-T6 with an elongated recognition domain inhibits HIV-1 production and has both the efficacy and specificity to be a promising candidate for HIV-1 gene therapy.

Small RNAs to treat human immunodeficiency virus type 1 infection by gene therapy.

Current drug therapies for human immunodeficiency virus type 1 (HIV) infection are effective in preventing progression to acquired immune deficiency syndrome but do not eliminate the infection and are associated with unwanted side effects. A potential alternative is to modify the genome of patient cells via gene therapy to confer HIV resistance to these cells. Small RNAs are the largest and most diverse group of anti-HIV genes that have been developed for engineering HIV resistant cells. In this review, we summarize progress on the three major classes of anti-HIV RNAs including short hairpin RNAs that use the RNA interference pathway, RNA decoys and aptamers that bind specifically to a protein or RNA as well as ribozymes that mediate cleavage of specific targets. We also review methods used for the delivery of these genes into the genome of patient cells and provide some perspectives on the future of small RNAs in HIV therapy.

[B-cell non-Hodgkin lymphoma discovery after observation of a platelet satellitism around atypical lymphocytes]. / Découverte d'un lymphome B non hodgkinien suite à l'observation d'un satellitisme plaquettaire autour de lymphocytes atypiques.

We report the case of an 83-year-old woman admitted to the accident and emergency unit for pneumopathy. The blood cell count on the DXH automaton (Coulter® ™ UniCel® DxH) found a normochromic and normocytic anemia, anormal platelet count and subnormal leukocyte formula. The only alarm raised by the automaton was the presence of a basocytosis. A control of the leucocyte count by flow cytometry and blood smear has been done. It revealed there was no basocytosis, but showed the presence of an atypical monomorphic lymphocytes B population (T/B < 1 ratio in flow cytometry), around which platelets aggregated. The lymphocytic immunophenotyping allowed us to highlight the presence of a CD5+ B clonal subpopulation.

ADAR1 and PKR, interferon stimulated genes with clashing effects on HIV-1 replication.

The induction of hundreds of Interferon Stimulated Genes (ISGs) subsequent to virus infection generates an antiviral state that functions to restrict virus growth at multiple steps of their replication cycles. In the context of Human Immunodeficiency Virus-1 (HIV-1), ISGs also possess antiviral functions, but some ISGs show proapoptotic or proviral activity. One of the most studied ISGs, the RNA activated Protein Kinase (PKR), shuts down the viral protein synthesis upon activation. HIV-1 has evolved to evade its inhibition by PKR through viral and cellular mechanisms. One of the cellular mechanisms is the induction of another ISG, the Adenosine Deaminase acting on RNA 1 (ADAR1). ADAR1 promotes viral replication by acting as an RNA sensing inhibitor, by editing viral RNA and by inhibiting PKR. This review challenges the orthodox dogma of ISGs as antiviral proteins, by demonstrating that two ISGs have opposing and clashing effects on viral replication.

Higher Cytopathic Effects of a Zika Virus Brazilian Isolate from Bahia Compared to a Canadian-Imported Thai Strain.

Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.

RNA Interference Therapies for an HIV-1 Functional Cure.

HIV-1 drug therapies can prevent disease progression but cannot eliminate HIV-1 viruses from an infected individual. While there is hope that elimination of HIV-1 can be achieved, several approaches to reach a functional cure (control of HIV-1 replication in the absence of drug therapy) are also under investigation. One of these approaches is the transplant of HIV-1 resistant cells expressing anti-HIV-1 RNAs, proteins or peptides. Small RNAs that use RNA interference pathways to target HIV-1 replication have emerged as competitive candidates for cell transplant therapy and have been included in all gene combinations that have so far entered clinical trials. Here, we review RNA interference pathways in mammalian cells and the design of therapeutic small RNAs that use these pathways to target pathogenic RNA sequences. Studies that have been performed to identify anti-HIV-1 RNA interference therapeutics are also reviewed and perspectives on their use in combination gene therapy to functionally cure HIV-1 infection are provided.

Inhibition of the inflammatory response to stress by targeting interaction between PKR and its cellular activator PACT.

PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.