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HIV-1 viral infectivity factor (Vif) alters processive single-stranded DNA scanning of the retroviral restriction factor APOBEC3G.

Feng Y, Love RP, Chelico L - J. Biol. Chem. (2013)

Bottom Line: Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation.Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G.That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (-)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of VifIIIB- and VifHXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

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A3G-induced mutagenesis is inhibited by VifHXB2 and VifIIIB in a model HIV-1 replication system.A–C, spectra of mutations incurred in the 368-nt prot-lacZα construct are plotted as the percentage of clones containing a mutation at a particular location (nt) for A3G (A), A3G in the presence of VifHXB2 (B), and A3G in the presence of VifIIIB (C). D–F, histograms were generated to illustrate the population distribution of mutations per prot-lacZα construct for A3G (D), A3G in the presence of VifHXB2 (E), and A3G in the presence of VifIIIB (F).
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Figure 1: A3G-induced mutagenesis is inhibited by VifHXB2 and VifIIIB in a model HIV-1 replication system.A–C, spectra of mutations incurred in the 368-nt prot-lacZα construct are plotted as the percentage of clones containing a mutation at a particular location (nt) for A3G (A), A3G in the presence of VifHXB2 (B), and A3G in the presence of VifIIIB (C). D–F, histograms were generated to illustrate the population distribution of mutations per prot-lacZα construct for A3G (D), A3G in the presence of VifHXB2 (E), and A3G in the presence of VifIIIB (F).

Mentions: A plot of the distribution of A3G-induced mutations recovered from the sequenced clones demonstrates a mutational gradient in the 5′ → 3′ orientation, meaning that deaminations were biased toward the 5′-end of the nascently synthesized cDNA (Fig. 1A). This has been observed previously for A3G in vivo due to HIV-1 replication dynamics in which (−)DNA furthest from the PPT is single-stranded the longest and incurs the most deaminations (7, 10, 11). To determine whether Vif could influence the deamination activity of A3G, we added Vif at excess (∼2-fold more than A3G). It can be seen by visual inspection of the mutation spectra that addition of VifIIIB or VifHXB2 caused a decrease in A3G-induced mutagenesis (Fig. 1A) with VifHXB2 (Fig. 1B) having a greater inhibitory effect than VifIIIB (Fig. 1C). The decrease in the A3G-induced mutations when VifHXB2 (Fig. 1B) or VifIIIB (Fig. 1C) was present was greater in the prot likely because of the shorter time that this region is single-stranded in comparison with the lacZα due to the proximity to the PPT. The clones were also binned for analysis based on the number of mutations per prot-lacZα (Fig. 1, D–F). A3G was able to induce a widespread number of mutations. We found a range of 3–12 mutations per prot-lacZα occurring for A3G (Fig. 1D). The data are far less spread when A3G-induced mutagenesis occurred in the presence of VifHXB2 where we found only three to five mutations per prot-lacZα ∼55% of the time and 0–2 mutations per prot-lacZα ∼35% of the time (Fig. 1E). For VifIIIB, the reduction in A3G-induced mutations is demonstrated by a shift of mutations down to three to eight mutations per prot-lacZα (Fig. 1F). The data were analyzed further by comparing the frequency of mutations in heavily mutated sites in the presence of A3G and the in absence and presence of Vif (Table 1). From this analysis, we found that VifHXB2 caused six of the eight heavily mutated sites to be mutated at a lower level (Table 1, A3G + VifHXB2). The detrimental effect of VifIIIB on A3G-induced mutagenesis was only significant in the prot region (Table 1, A3G + VifIIIB).


HIV-1 viral infectivity factor (Vif) alters processive single-stranded DNA scanning of the retroviral restriction factor APOBEC3G.

Feng Y, Love RP, Chelico L - J. Biol. Chem. (2013)

A3G-induced mutagenesis is inhibited by VifHXB2 and VifIIIB in a model HIV-1 replication system.A–C, spectra of mutations incurred in the 368-nt prot-lacZα construct are plotted as the percentage of clones containing a mutation at a particular location (nt) for A3G (A), A3G in the presence of VifHXB2 (B), and A3G in the presence of VifIIIB (C). D–F, histograms were generated to illustrate the population distribution of mutations per prot-lacZα construct for A3G (D), A3G in the presence of VifHXB2 (E), and A3G in the presence of VifIIIB (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585047&req=5

Figure 1: A3G-induced mutagenesis is inhibited by VifHXB2 and VifIIIB in a model HIV-1 replication system.A–C, spectra of mutations incurred in the 368-nt prot-lacZα construct are plotted as the percentage of clones containing a mutation at a particular location (nt) for A3G (A), A3G in the presence of VifHXB2 (B), and A3G in the presence of VifIIIB (C). D–F, histograms were generated to illustrate the population distribution of mutations per prot-lacZα construct for A3G (D), A3G in the presence of VifHXB2 (E), and A3G in the presence of VifIIIB (F).
Mentions: A plot of the distribution of A3G-induced mutations recovered from the sequenced clones demonstrates a mutational gradient in the 5′ → 3′ orientation, meaning that deaminations were biased toward the 5′-end of the nascently synthesized cDNA (Fig. 1A). This has been observed previously for A3G in vivo due to HIV-1 replication dynamics in which (−)DNA furthest from the PPT is single-stranded the longest and incurs the most deaminations (7, 10, 11). To determine whether Vif could influence the deamination activity of A3G, we added Vif at excess (∼2-fold more than A3G). It can be seen by visual inspection of the mutation spectra that addition of VifIIIB or VifHXB2 caused a decrease in A3G-induced mutagenesis (Fig. 1A) with VifHXB2 (Fig. 1B) having a greater inhibitory effect than VifIIIB (Fig. 1C). The decrease in the A3G-induced mutations when VifHXB2 (Fig. 1B) or VifIIIB (Fig. 1C) was present was greater in the prot likely because of the shorter time that this region is single-stranded in comparison with the lacZα due to the proximity to the PPT. The clones were also binned for analysis based on the number of mutations per prot-lacZα (Fig. 1, D–F). A3G was able to induce a widespread number of mutations. We found a range of 3–12 mutations per prot-lacZα occurring for A3G (Fig. 1D). The data are far less spread when A3G-induced mutagenesis occurred in the presence of VifHXB2 where we found only three to five mutations per prot-lacZα ∼55% of the time and 0–2 mutations per prot-lacZα ∼35% of the time (Fig. 1E). For VifIIIB, the reduction in A3G-induced mutations is demonstrated by a shift of mutations down to three to eight mutations per prot-lacZα (Fig. 1F). The data were analyzed further by comparing the frequency of mutations in heavily mutated sites in the presence of A3G and the in absence and presence of Vif (Table 1). From this analysis, we found that VifHXB2 caused six of the eight heavily mutated sites to be mutated at a lower level (Table 1, A3G + VifHXB2). The detrimental effect of VifIIIB on A3G-induced mutagenesis was only significant in the prot region (Table 1, A3G + VifIIIB).

Bottom Line: Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation.Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G.That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (-)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of VifIIIB- and VifHXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

Show MeSH
Related in: MedlinePlus