Human APOBEC3G can restrict retroviral infection in avian cells and acts independently of both UNG and SMUG1 - PubMed (original) (raw)

Human APOBEC3G can restrict retroviral infection in avian cells and acts independently of both UNG and SMUG1

Marc-André Langlois et al. J Virol. 2008 May.

Abstract

APOBEC3 proteins are mammal-specific cytidine deaminases that can restrict retroviral infection. The exact mechanism of the restriction remains unresolved, but one model envisions that uracilated retroviral cDNA, generated by cytidine deamination, is the target of cellular glycosylases. While restriction is unaffected by UNG deficiency, it has been suggested that the SMUG1 glycosylase might provide a backup. We found that retroviral restriction can be achieved by introducing human APOBEC3G into chicken cells (consistent with the components necessary for APOBEC3-mediated restriction predating mammalian evolution) and used this assay to show that APOBEC3G-mediated restriction can occur in cells deficient in both UNG and SMUG1.

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Figures

FIG. 1.

FIG. 1.

APOBEC3G-mediated restriction of HIV (ΔVif) in human cells is independent of UNG. (A) Titration of transiently transfected Flag-tagged APOBEC3G (A3G), APOBEC3F (A3F), and a catalytically inactivated mutant of APOBEC3G (A3Gmut) in 293T cells, comparing their potencies in restricting HIV (ΔVif) infection. APOBEC3 expression plasmids (28), the HIV (ΔVif) vector p8.91 expressing the eGFP reporter (36), and viral infectivity assays in 293T cells are described elsewhere (28). A3Gmut has a cysteine-to-serine substitution at amino acid 288 that has been previously shown to compromise both deaminase and antiviral activities (13, 32, 34, 35, 40, 45, 53). (B) Infection assay showing that mutation load does not directly correlate with the level of inhibition of HIV (ΔVif) infection. Ctrl, control plasmid. Error bars indicate standard deviations. (C) Infection assay in 293T cells, showing that transient transfection of Ugi in A3G-expressing cells has no significant effect on HIV (ΔVif) restriction. (D) Oligonucleotide assay showing glycosylase activities of UNG and SMUG1 in 293T cell extracts. S indicates the uncleaved oligonucleotide substrate and P the cleaved product. Detailed procedures for the oligonucleotide assays were described previously (11). The amount of extract was such that the UNG activity in the control sample (lane 1) was sufficient to saturate the assay, shifting all the substrate to product.

FIG. 2.

FIG. 2.

UNG-independent inhibition of RSV by human APOBEC3G. (A) DF1 stable transfectants expressing A3G and/or Ugi were infected with replicative RSV-eGFP as described previously (41). Viral supernatants were then used to infect DF1 cells, and infectivity was assessed according to the percentage of cells expressing eGFP relative to the control after 24 h. eGFP expression was detectable in 95% of control (Ctrl) cells after 24 h (data not shown). APOBEC3G had no effect on viral infectivity when expressed in the target cells (data not shown). SMUG1mut contains an inactivating mutation in the catalytic site (12). Error bars indicate standard deviations. (B) Top, Western blotting performed on cell lysates, depicting expression of empty vector (Ctrl), Ugi, Flag-tagged APOBEC3G (A3G), and Flag-tagged APOBEC3G and Ugi (A3G-Ugi) stably transfected in DF1 cells. Bottom, encapsidation of Flag-tagged APOBEC3G into RSV particles. Encapsidation assays were performed as previously described (28). (C) Oligonucleotide assay performed on DF1-Ugi whole-cell extracts. The methods and oligonucleotide pair used were identical to those for Fig. 1D. UNG activity was tested on a double-stranded oligonucleotide with a central U·A pair and a fluorescein marker on the 3′ end of the U-containing oligonucleotide. S indicates the uncleaved oligonucleotide substrate and P the cleaved product.

References

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