Discovery of the RNA interference (RNAi) pathway has led to exciting new strategies for developing HIV treatment. This study was to find out the highly effective and conserved siRNA target sequences for improving RNAi-based therapy against the HIV-1. We constructed 30 shRNA expression plasmids for expressing different siRNAs targeted to HIV-1 vif and co-transfected them with the pNL4-3 to score for its ability to inhibit the expression of p24 protein of HIV-1. Then, the highly effective siRNAs targeting sequences were selected to align with 625 HIV-1 sequences in database including all HIV-1 subtypes to ana lyze their conserved character. In addition, vif37 the highly effective and most conserved target sequence was confirmed of its sequence-specific inhibition by independent reporter assays. MT-4 cell transduced with lentiviral shRNA-vif37 vector could inhibit HIV-1(NL4.3) replication in vitro. Moreover, MT-4-vif37 cloned from transduced MT-4 cell could stably express shRNA-vif37 and inhibit virus replication more efficiently when challenged with high titer virus. These results showed that RNAi has great potential as an antiviral gene therapy approach and supports the efforts to develop treatment for HIV-1-infected individuals.
Acquired Immunodeficiency Syndrome ; therapy ; Base Sequence ; Humans ; Lentivirus ; genetics ; Molecular Sequence Data ; RNA Interference ; RNA, Small Interfering ; chemistry ; genetics ; Virus Replication ; vif Gene Products, Human Immunodeficiency Virus ; antagonists & inhibitors ; genetics

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3 protein G (APOBEC3G) is part of the innate immune system of host cells and has cytidine deaminase activity. It specifically incorporates into the virion during HIV-1 replication. The incorporation of APOBEC3G needs its interaction with HIV-1 Gag. In the HIV-1 reverse transcription process, APOBEC3G deaminates dC to dU in the first minus strand cDNA, and then induces extensive hypermutation in the viral genome. Besides deamination, APOBEC3G also inhibits HIV-1 by some kinds of non-deamination mechanisms which need to be further elucidated. HIV-1 Vif counteracts the activity of APOBEC3G by an ubiquitin-proteasome-mediated degradation of APOBEC3G. As a broad spectrum inhibitor of viruses, APOBEC3G also inhibits various retroviruses, retrotransposons and other viruses like HBV. Upregulating the expression of APOBEC3G or blocking the Vif-mediated degradation of APOBEC3G might be novel strategies to treat HIV-1 infection in the future.
APOBEC-3G Deaminase ; Amino Acid Substitution ; Anti-HIV Agents ; metabolism ; Cytidine Deaminase ; genetics ; metabolism ; Gene Expression ; HIV Infections ; metabolism ; HIV-1 ; genetics ; physiology ; Humans ; Virus Replication ; vif Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

APOBEC3 is a class of cytidine deaminase, which is considered as a new member of the innate immune system, and APOBEC3G belongs to this family. The research about APOBEC3G is a new direction of innate immune defense mechanism against virus. APOBEC3G has the restrictive activity on many viral replications, which deaminates dC to dU in the viral genome and then induces extensive hypermutation. APOBEC3G can also interrupt viral replication at several phases such as reverse transcription, replication, nucleocapsid and so on by non-deamination mechanisms. However, virus can encode viral proteins to counteract the restriction activity of APOBEC3G. Elucidation of the antagonistic interaction between APOBEC3G and the virus will be contributed to development of new antiviral drugs in the future.
APOBEC-3G Deaminase ; Animals ; Cytidine Deaminase ; genetics ; metabolism ; DNA Replication ; Deamination ; HIV-1 ; physiology ; Hepacivirus ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Humans ; Paramyxoviridae ; genetics ; physiology ; Retroviridae ; physiology ; Virus Replication ; vif Gene Products, Human Immunodeficiency Virus ; metabolism

Human immunodeficiency virus type 1 (HIV-1) viral infectivity factor (Vif), one of the accessory proteins, which is a small basic phosphoprotein, is essential for viral replication and pathogenesis. The best well-characterized function of Vif is its ability to neutralize the host cell antiviral factor, apolipoprotein B mRNA editing enzyme catalytic polypeptide like 3G (APOBEC3G), which makes the viral particles more infective. In addition, Vif can regulate the reverse transcription and the advanced stage of replication of the virus particle, as well as induce the termination of cell cycle at G2 stage and so on. The designed drug aimed directly at Vif can efficiently block the maturation and infectivity of HIV-1. In this review, the structure, function and especially the related inhibitors of Vif are reviewed.
APOBEC-3G Deaminase ; Amino Acid Sequence ; Anti-HIV Agents ; pharmacology ; Cytidine Deaminase ; metabolism ; Ethylenediamines ; pharmacology ; HIV-1 ; physiology ; Humans ; Reverse Transcription ; Virus Replication ; vif Gene Products, Human Immunodeficiency Virus ; antagonists & inhibitors ; genetics ; metabolism ; physiology

Effective and specific RNA interference (RNAi) elements are essential for the RNAi-based anti-HIV-1 research which has achieved extensive application. vif37 targeted to HIV-1 vi f is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase II promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37 H. The replication of HIV-1 was inhibited in MT-4-miR37 H cells which could express miR-vif37 H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37 H expression element. Real-time RT-PCR revealed that miR-vif37 H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not effected by the expression of miR-vif37 H in MT-4-miR37 H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.
Cell Line ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; metabolism ; HIV Infections ; virology ; HIV-1 ; genetics ; physiology ; Humans ; Lentivirus ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Nucleic Acid Conformation ; RNA Interference ; vif Gene Products, Human Immunodeficiency Virus ; chemistry ; genetics ; metabolism

The mammalian APOBEC3G protein (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3 protein G, APOBEC3G) is an important component of the cellular innate immune response to retroviral infection. APOBEC3G can extinguish HIV-1 (human immunodeficiency virus type 1) infectivity by its incorporation into virus particles and subsequent cytosine deaminase activity to block replication of HIV-1. HIV-1 Vif (viral infectivity factor) suppresses various APOBEC3 proteins through a common mechanism which induces the degradation of target proteins. Therefore, the interrelation of Vif-APOBEC3G has been extensively studied, which represents attractive targets for the development of novel inhibitors. We summarize the papers in which the detection technique and methods have been developed to assay the anti-HIV activity and its mechanism, such as western-blotting, co-immunoprecipitation, pulse-chase experiments, bioluminescence resonance energy transfer, biomolecular interaction analysis. This review is towards developing therapeutics aimed at the Vif-APOBEC3G axis.
APOBEC-3G Deaminase ; Anti-HIV Agents ; pharmacology ; Blotting, Western ; Cytidine Deaminase ; antagonists & inhibitors ; Fluorescence ; HIV-1 ; drug effects ; Immunoprecipitation ; Surface Plasmon Resonance ; vif Gene Products, Human Immunodeficiency Virus ; antagonists & inhibitors