RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but—in contrast to what is observed with nef—no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.

Human Immunodeficiency Virus Type 1 Escape Is Restricted When Conserved Genome Sequences Are Targeted by RNA Interference

Bifidobacteria are considered one of the most beneficial probiotics and have been widely studied for their effects in preventing and treating specific pathological conditions. The present study explored the antiviral activity of Bifidobacterium adolescentis SPM0212 isolated from healthy Koreans against hepatitis B virus (HBV) and its mechanism of action. To determine the effect of B. adolescentis SPM0212 against HBV, the level of HBV surface antigen (HBsAg) in the culture medium and the levels of viral transcripts in HepG2.2.15 cells were measured by enzyme-linked immunosorbent assay and reverse transcription-quantitative PCR (RT-qPCR), respectively. To clarify the mechanism, we performed RT-qPCR using specific primers for genes encoding Interferon (IFN)-signaling components and IFN-inducible antiviral effectors. The cell extract of B. adolescentis SPM0212 dose-dependently decreased the extracellular HBsAg level by up to 50 %. Its gene expression in HepG2.2.15 cells was also inhibited by 40 %. This extract significantly increased the expression level of myxovirus resistance A, which is an IFN-inducible antiviral effector. Furthermore, the antiviral activity was observed in the fraction of compound(s) with molecular weights under 30 kDa. Thus, the cell extract of B. adolescentis SPM0212 inhibits HBV and its antiviral mechanism is associated with the Mx GTPase pathway.

Antiviral activity of Bifidobacterium adolescentis SPM0212 against Hepatitis B virus

Background

RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences.



Methods

In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay.



Results

Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study.



Conclusions

These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs. Copyright © 2009 John Wiley & Sons, Ltd.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jgm.1329

Stringent testing identifies highly potent and escape‐proof anti‐HIV short hairpin RNAs

Bioactivity evaluations of ingredients extracted from the flowers of Citrus aurantium L. var. amara Engl.

RNA interference (RNAi) has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs) against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study. An artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed. The strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.

/pdf/efficient-inhibition-of-hiv-1-replication-by-an-artificial-4po4k8gc22.pdf

Zhang

Papers

Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus de