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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 D128K-induced mutagenesis is unaffected 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 D128K (A), D128K in the presence of VifHXB2 (B), and D128K in the presence of VifIIIB (C). D–F, histograms illustrate that the population distribution of mutations per prot-lacZα construct for D128K (D) was not decreased in the presence of VifHXB2 (E) or VifIIIB (F).
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Figure 5: A3G D128K-induced mutagenesis is unaffected 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 D128K (A), D128K in the presence of VifHXB2 (B), and D128K in the presence of VifIIIB (C). D–F, histograms illustrate that the population distribution of mutations per prot-lacZα construct for D128K (D) was not decreased in the presence of VifHXB2 (E) or VifIIIB (F).

Mentions: We tested the ability of D128K to induce mutations in a model HIV-1 replication system in the absence and presence of the Vif variants. The mutational spectrum for D128K alone shows peaks for highly mutated sites in the same locations as for A3G (compare Fig. 1A and Fig. 5A). The intensity of the mutations among the sites differed slightly likely because of the stochastic nature of A3G deamination (18). The mutation frequencies are nearly identical for D128K and A3G at the population level (D128K, 0.96 (Table 4); A3G, 0.87 (Table 2)) and clone level (D128K, 2.2 × 10−2 (Table 4); A3G, 2.2 × 10−2 (Table 2)). Overall, the results are consistent with D128K behaving similarly to A3G in this assay. When both VifIIIB and Vif HXB2 were added to the reaction with D128K, we observed no decrease in the mutagenic ability of D128K (Fig. 5, A–F, and Table 4) in contrast to A3G (Fig. 1, A–F, and Table 2). In addition, we found no effects on D128K processive scanning by sliding by the addition of VifHXB2 or VifIIIB to the model HIV-1 replication assay as the mutation cluster frequency (Fig. 6) and frequency of mutations (Fig. 5 and Table 4) remained constant in all three experimental conditions. This observation is in agreement with data obtained on the 118-nt substrate where neither VifHXB2 nor VifIIIB was found to change D128K processivity (data not shown). Altogether, the data support a model in which an alteration in the processivity of A3G in the presence of Vif is caused by a Vif-A3G interaction.


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 D128K-induced mutagenesis is unaffected 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 D128K (A), D128K in the presence of VifHXB2 (B), and D128K in the presence of VifIIIB (C). D–F, histograms illustrate that the population distribution of mutations per prot-lacZα construct for D128K (D) was not decreased in the presence of VifHXB2 (E) or VifIIIB (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: A3G D128K-induced mutagenesis is unaffected 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 D128K (A), D128K in the presence of VifHXB2 (B), and D128K in the presence of VifIIIB (C). D–F, histograms illustrate that the population distribution of mutations per prot-lacZα construct for D128K (D) was not decreased in the presence of VifHXB2 (E) or VifIIIB (F).
Mentions: We tested the ability of D128K to induce mutations in a model HIV-1 replication system in the absence and presence of the Vif variants. The mutational spectrum for D128K alone shows peaks for highly mutated sites in the same locations as for A3G (compare Fig. 1A and Fig. 5A). The intensity of the mutations among the sites differed slightly likely because of the stochastic nature of A3G deamination (18). The mutation frequencies are nearly identical for D128K and A3G at the population level (D128K, 0.96 (Table 4); A3G, 0.87 (Table 2)) and clone level (D128K, 2.2 × 10−2 (Table 4); A3G, 2.2 × 10−2 (Table 2)). Overall, the results are consistent with D128K behaving similarly to A3G in this assay. When both VifIIIB and Vif HXB2 were added to the reaction with D128K, we observed no decrease in the mutagenic ability of D128K (Fig. 5, A–F, and Table 4) in contrast to A3G (Fig. 1, A–F, and Table 2). In addition, we found no effects on D128K processive scanning by sliding by the addition of VifHXB2 or VifIIIB to the model HIV-1 replication assay as the mutation cluster frequency (Fig. 6) and frequency of mutations (Fig. 5 and Table 4) remained constant in all three experimental conditions. This observation is in agreement with data obtained on the 118-nt substrate where neither VifHXB2 nor VifIIIB was found to change D128K processivity (data not shown). Altogether, the data support a model in which an alteration in the processivity of A3G in the presence of Vif is caused by a Vif-A3G interaction.

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