14 research outputs found
Inhibition of HIV-1 expression and replication by SOFA-HDV ribozymes against Tat and Rev mRNA sequences
RIG-I Is Required for the Inhibition of Measles Virus by Retinoids
Vitamin A can significantly decrease measles-associated morbidity and mortality. Vitamin A can inhibit the replication of measles virus (MeV) in vitro through an RARα- and type I interferon (IFN)-dependent mechanism. Retinoid-induced gene I (RIG-I) expression is induced by retinoids, activated by MeV RNA and is important for IFN signaling. We hypothesized that RIG-I is central to retinoid-mediated inhibition of MeV in vitro. We demonstrate that RIG-I expression is increased in cells treated with retinoids and infected with MeV. The central role of RIG-I in the retinoid-anti-MeV effect was demonstrated in the Huh-7/7.5 model; the latter cells having non-functional RIG-I. RAR-dependent retinoid signaling was required for the induction of RIG-I by retinoids and MeV. Retinoid signaling was also found to act in combination with IFN to induce high levels of RIG-I expression. RIG-I promoter activation required both retinoids and MeV, as indicated by markers of active chromatin. IRF-1 is known to be regulated by retinoids and MeV, but we found recruitment of IRF-1 to the RIG-I promoter by retinoids alone. Using luciferase expression constructs, we further demonstrated that the IRF-1 response element of RIG-I was required for RIG-I activation by retinoids or IFN. These results reveal that retinoid treatment and MeV infection induces significant RIG-I. RIG-I is required for the retinoid-MeV antiviral response. The induction is dependent on IFN, retinoids and IRF-1
In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication.
International audienceRNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy
In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication
Activation of RIG-I promoter only upon combination of MeV infection and retinoid treatment.
<p>U937 cells were infected with MeV at an MOI of 0.1 and/or treated with 1 µM ATRA or DMSO for 24 hours. 1000 U/ml of IFNβ was used as a positive control. These samples were then immunoprecipitated the following primary antibodies (A) Acetylate Histone H3 (B) Pol II (C) IRF-1. The pulled-down DNA was analyzed by qPCR using primers specific for the RIG-I promoter as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022323#s4" target="_blank">materials and methods</a>. Data presented are representative of experiments performed in triplicate between two and three times (N = 2–3). *p<0.05, **p<0.01, ***p<0.001.</p
MeV with ATRA induces the soluble factor IFN to elicit the expression of RIG-I.
<p>(A) Cells were infected with MeV at an MOI of 0.1 in the presence of 1 µM ATRA or DMSO, and either IFNαβ-receptor blocking antibodies or isotype control. RNA was extracted at 24 h and RIG-I expression was measured by qPCR. Data presented are representative of 2 experiments performed in triplicate (N = 2). (B) Transwell membrane inserts with 0.02 µm pores were used to separate the infected cells from the uninfected, bystander cells in the inner chamber <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022323#pone.0022323-Trottier2" target="_blank">[9]</a>. (C) Cells from transwell-free control wells and the inner chamber bystander cells were harvested after 48 hours and RIG-I mRNA was measured by qPCR. Data presented are representative of three experiments performed in triplicate (N = 3). (D) Supernatants from the control wells and the inner chambers of the transwells were used to treat fresh U937 cells with either IFNαβ-receptor blocking antibody or isotype control antibody. Following 24 hours of incubation, RIG-I expression was assessed by qPCR. Data presented are representative of three experiments performed in triplicate (N = 3). **p<0.01, ***p<0.001.</p
Retinoic acid nuclear receptors bind to the RIG-I promoter.
<p>(A) Diagram of the RIG-I promoter showing the known IRF1 binding site. Arrows represent the site of primers used in ChIP experiments. (B) RARα and RXR were immunoprecipitated from cells treated with 1 µM ATRA or DMSO (N = 2) (C) U937 cells were infected with MeV at an MOI of 0.1 and/or treated with 1 µM ATRA or DMSO for 24 hours. 1000 U/ml of IFNβ was used as a positive control. These samples were then immunoprecipitated RARα primary antibodies. The pulled-down DNA was analyzed by qPCR using primers specific for the RIG-I promoter as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022323#s4" target="_blank">materials and methods</a>. Data presented are representative of three experiments performed in triplicate (N = 3). *p<0.05, **p<0.01, ***p<0.001.</p