HIV-1 Vif, APOBEC, and Intrinsic Immunity

Members of the APOBEC family of cellular cytidine deaminases represent a recently identified group of proteins that provide immunity to infection by retroviruses and protect the cell from endogenous mobile retroelements. Yet, HIV-1 is largely immune to the intrinsic antiviral effects of APOBEC proteins because it encodes Vif (viral infectivity factor), an accessory protein that is critical for in vivo replication of HIV-1. In the absence of Vif, APOBEC proteins are encapsidated by budding virus particles and either cause extensive cytidine to uridine editing of negative sense single-stranded DNA during reverse transcription or restrict virus replication through deaminase-independent mechanisms. Thus, the primary function of Vif is to prevent encapsidation of APOBEC proteins into viral particles. This is in part accomplished by the ability of Vif to induce the ubiquitin-dependent degradation of some of the APOBEC proteins. However, Vif is also able to prevent encapsidation of APOBEC3G and APOBEC3F through degradation-independent mechanism(s). The goal of this review is to recapitulate current knowledge of the functional interaction of HIV-1 and its Vif protein with the APOBEC3 subfamily of proteins and to summarize our present understanding of the mechanism of APOBEC3-dependent retrovirus restriction

Analysis of the contribution of cellular and viral RNA to the packaging of APOBEC3G into HIV-1 virions

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Targeted cleavage of HIV-1 envelope gene by a DNA enzyme and inhibition of HIV-1 envelope-CD4 mediated cell fusion

AbstractWith the ultimate aim of developing an effective antiviral strategy against HIV-1, a mono-DNA enzyme possessing the 10–23 catalytic motif [Santoro and Joyce (1997) Proc. Natl. Acad. Sci. USA 94, 4264–4266] was synthesized against the HIV-1 envelope gene. We tested the in vitro cleavage efficiency of the 178 bp long truncated HIV-1 Env transcript by DNA enzyme 6339. Protein independent and Mg2+ dependent specific cleavage products were obtained. As soon as 5 min after mixing equimolar concentrations of DNA enzyme and substrate RNA, more than 50% cleavage was observed which increased steadily over a period of 4 h. Very little cleavage was obtained at 1 mM MgCl2 concentration which improved significantly when the concentration of MgCl2 was increased up to 20 mM. Specific inhibition of cell membrane fusion caused by the interaction of gp160 and CD4 in HeLa cells was observed when the above DNA enzyme was used. Thus, these chemically synthesized DNA enzymes could prove to be very useful for in vivo application

Encapsidation of APOBEC3G into HIV-1 virions involves lipid raft association and does not correlate with APOBEC3G oligomerization

<p>Abstract</p> <p>Background</p> <p>The cellular cytidine deaminase APOBEC3G (A3G), when incorporated into the human immunodeficiency virus type 1 (HIV-1), renders viral particles non-infectious. We previously observed that mutation of a single cysteine residue of A3G (C100S) inhibited A3G packaging. In addition, several recent studies showed that mutation of tryptophan 127 (W127) and tyrosine 124 (Y124) inhibited A3G encapsidation suggesting that the N-terminal CDA constitutes a viral packaging signal in A3G. It was also reported that W127 and Y124 affect A3G oligomerization.</p> <p>Results</p> <p>Here we studied the mechanistic basis of the packaging defect of A3G W127A and Y124A mutants. Interestingly, cell fractionation studies revealed a strong correlation between encapsidation, lipid raft association, and genomic RNA binding of A3G. Surprisingly, the presence of a C-terminal epitope tag affected lipid raft association and encapsidation of the A3G W127A mutant but had no effect on wt A3G encapsidation, lipid raft association, and interaction with viral genomic RNA. Mutation of Y124 abolished A3G encapsidation irrespective of the presence or absence of an epitope tag. Contrasting a recent report, our co-immunoprecipitation studies failed to reveal a correlation between A3G oligomerization and A3G encapsidation. In fact, our W127A and Y124A mutants both retained the ability to oligomerize.</p> <p>Conclusion</p> <p>Our results confirm that W127 and Y124 residues in A3G are important for encapsidation into HIV-1 virions and our data establish a novel correlation between genomic RNA binding, lipid raft association, and viral packaging of A3G. In contrast, we were unable to confirm a role of W127 and Y124 in A3G oligomerization and we thus failed to confirm a correlation between A3G oligomerization and virus encapsidation.</p

Sequence specific cleavage of the HIV-1 coreceptor CCR5 gene by a hammer-head ribozyme and a DNA-enzyme: inhibition of the coreceptor function by DNA-enzyme

AbstractThe chemokine receptor CCR5 is used as a major coreceptor for fusion and entry by non-syncytia inducing macrophage tropic isolates of HIV-1, which is mainly involved in transmission. Individuals who are homozygous for the Δ32 allele of CCR5 are usually resistant to HIV-1 infection and continue to lead a normal healthy life. Thus this gene is dispensable and is, therefore, an attractive target in the host cell for interfering specifically with the virus-host interaction. With the aim to develop a specific antiviral approach at the molecular level, we have synthesized a hammer-head ribozyme and a DNA-enzyme. Both ribozyme and DNA-enzyme cleaved the CCR5 RNA in a sequence specific manner. This cleavage was protein independent but Mg2+ dependent. The extent of cleavage increased with increasing concentration of magnesium chloride. DNA-enzyme was more effective in cleaving a full length (1376 bases) in vitro generated transcript of CCR5 gene. In this communication, we show that the DNA-enzyme when introduced into a mammalian cell, results in decreased CD4-CCR5-gp160 mediated fusion of cell membranes. Potential applications of these trans acting molecules are discussed

Differential Sensitivity of “Old” versus “New” APOBEC3G to Human Immunodeficiency Virus Type 1 Vif▿

HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inhibiting its encapsidation into virions. Here, we compared the relative sensitivity to Vif of APOBEC3G in stable HeLa cells containing APOBEC3G (HeLa-A3G cells) versus that of newly synthesized APOBEC3G. We observed that newly synthesized APOBEC3G was more sensitive to degradation than preexisting APOBEC3G. Nevertheless, preexisting and transiently expressed APOBEC3G were packaged with similar efficiencies into vif-deficient human immunodeficiency virus type 1 (HIV-1) virions, and Vif inhibited the encapsidation of both forms of APOBEC3G into HIV particles equally well. Our results suggest that HIV-1 Vif preferentially induces degradation of newly synthesized APOBEC3G but indiscriminately inhibits encapsidation of “old” and “new” APOBEC3G