Limits...
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.

Show MeSH

Related in: MedlinePlus

Processivity of A3G in the absence and presence of VifHXB2 and VifIIIB.A, deamination of a 118-nt F-labeled ssDNA substrate by A3G. Two CCC motifs within the ssDNA sequence are spaced 61 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 63-nt labeled fragment (5′-C and 3′-C). A3G processivity was decreased by ∼2-fold in the presence of VifHXB2 (+VifHXB2) and VifIIIB (+VifIIIB). B, deamination of the substrate described for A but with a 20-nt ssDNA annealed between the two CCC motifs that can block the sliding component of A3G processivity. This resulted in a 2-fold decrease in A3G processivity. Processive deaminations were still observed due to the jumping component of A3G processive movement. In the presence of VifHXB2, the processivity of A3G was nearly absent, indicating that the jumping component of A3G has been inhibited. This is in contrast to the VifIIIB, which had no effect on A3G jumping movement. C, deamination of the substrate described for B but with a 20-nt RNA annealed between the two CCC motifs. Processive scanning of A3G on this substrate in the absence and presence of VifHXB2 and VifIIIB was similar to that observed for B. The measurements of processivity (Processivity factor) are shown below the gel. Values are an average from at least three independent experiments, and the S.D. for the processivity factors are as follows: in A, A3G, 1.90; +VifHXB2, 0.03; +VifIIIB, 0.65; in B, A3G, 0.80; +VifHXB2, 0.20; +VifIIIB, 0.76; in C, A3G, 1.20; +VifHXB2, 0.16; +VifIIIB, 0.40.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585047&req=5

Figure 3: Processivity of A3G in the absence and presence of VifHXB2 and VifIIIB.A, deamination of a 118-nt F-labeled ssDNA substrate by A3G. Two CCC motifs within the ssDNA sequence are spaced 61 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 63-nt labeled fragment (5′-C and 3′-C). A3G processivity was decreased by ∼2-fold in the presence of VifHXB2 (+VifHXB2) and VifIIIB (+VifIIIB). B, deamination of the substrate described for A but with a 20-nt ssDNA annealed between the two CCC motifs that can block the sliding component of A3G processivity. This resulted in a 2-fold decrease in A3G processivity. Processive deaminations were still observed due to the jumping component of A3G processive movement. In the presence of VifHXB2, the processivity of A3G was nearly absent, indicating that the jumping component of A3G has been inhibited. This is in contrast to the VifIIIB, which had no effect on A3G jumping movement. C, deamination of the substrate described for B but with a 20-nt RNA annealed between the two CCC motifs. Processive scanning of A3G on this substrate in the absence and presence of VifHXB2 and VifIIIB was similar to that observed for B. The measurements of processivity (Processivity factor) are shown below the gel. Values are an average from at least three independent experiments, and the S.D. for the processivity factors are as follows: in A, A3G, 1.90; +VifHXB2, 0.03; +VifIIIB, 0.65; in B, A3G, 0.80; +VifHXB2, 0.20; +VifIIIB, 0.76; in C, A3G, 1.20; +VifHXB2, 0.16; +VifIIIB, 0.40.

Mentions: To investigate how VifIIIB and VifHXB2 inhibit A3G-induced mutagenesis and why these variants differ in their effect (Fig. 1 and Tables 1 and 2), we conducted assays measuring A3G-catalyzed deaminations on synthetic DNA substrates. Specifically, the assay can measure the processive scanning behavior of A3G by using a 118-nt ssDNA substrate with two 5′-CCC deamination motifs spaced 61 nt apart (Fig. 3A, schematic). Fig. 3A shows a characteristic result of A3G in this assay. A3G deaminated both the 5′- and 3′-proximal CCC motifs, but there was a 2-fold preference for the 5′-motif due to an inherent catalytic orientation specificity of the active site (22). Of importance is the presence of a double deamination band, which indicates that a deamination occurred at both the 5′-C and 3′-C on the same ssDNA (Fig. 3A, 63 nt). The processivity factor is calculated as a ratio of the intensity of this band to the calculated expected value of deaminations that would occur at both 5′- and 3′-proximal motifs if the enzyme were not processive (see “Experimental Procedures”). The processivity factor of 9.1 (Fig. 3A, below gel) means that A3G is 9 times more likely to undergo a processive deamination of both CCC motifs than to deaminate each of the motifs in separate enzyme-ssDNA encounters. VifHXB2 and VifIIIB were able to decrease the processivity of A3G by at least 1.5-fold from 9.1 to 4.1 or 5.6, respectively (Fig. 3A).


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)

Processivity of A3G in the absence and presence of VifHXB2 and VifIIIB.A, deamination of a 118-nt F-labeled ssDNA substrate by A3G. Two CCC motifs within the ssDNA sequence are spaced 61 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 63-nt labeled fragment (5′-C and 3′-C). A3G processivity was decreased by ∼2-fold in the presence of VifHXB2 (+VifHXB2) and VifIIIB (+VifIIIB). B, deamination of the substrate described for A but with a 20-nt ssDNA annealed between the two CCC motifs that can block the sliding component of A3G processivity. This resulted in a 2-fold decrease in A3G processivity. Processive deaminations were still observed due to the jumping component of A3G processive movement. In the presence of VifHXB2, the processivity of A3G was nearly absent, indicating that the jumping component of A3G has been inhibited. This is in contrast to the VifIIIB, which had no effect on A3G jumping movement. C, deamination of the substrate described for B but with a 20-nt RNA annealed between the two CCC motifs. Processive scanning of A3G on this substrate in the absence and presence of VifHXB2 and VifIIIB was similar to that observed for B. The measurements of processivity (Processivity factor) are shown below the gel. Values are an average from at least three independent experiments, and the S.D. for the processivity factors are as follows: in A, A3G, 1.90; +VifHXB2, 0.03; +VifIIIB, 0.65; in B, A3G, 0.80; +VifHXB2, 0.20; +VifIIIB, 0.76; in C, A3G, 1.20; +VifHXB2, 0.16; +VifIIIB, 0.40.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Processivity of A3G in the absence and presence of VifHXB2 and VifIIIB.A, deamination of a 118-nt F-labeled ssDNA substrate by A3G. Two CCC motifs within the ssDNA sequence are spaced 61 nt apart. Single deaminations of the 5′-C and 3′-C were detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule resulted in a 63-nt labeled fragment (5′-C and 3′-C). A3G processivity was decreased by ∼2-fold in the presence of VifHXB2 (+VifHXB2) and VifIIIB (+VifIIIB). B, deamination of the substrate described for A but with a 20-nt ssDNA annealed between the two CCC motifs that can block the sliding component of A3G processivity. This resulted in a 2-fold decrease in A3G processivity. Processive deaminations were still observed due to the jumping component of A3G processive movement. In the presence of VifHXB2, the processivity of A3G was nearly absent, indicating that the jumping component of A3G has been inhibited. This is in contrast to the VifIIIB, which had no effect on A3G jumping movement. C, deamination of the substrate described for B but with a 20-nt RNA annealed between the two CCC motifs. Processive scanning of A3G on this substrate in the absence and presence of VifHXB2 and VifIIIB was similar to that observed for B. The measurements of processivity (Processivity factor) are shown below the gel. Values are an average from at least three independent experiments, and the S.D. for the processivity factors are as follows: in A, A3G, 1.90; +VifHXB2, 0.03; +VifIIIB, 0.65; in B, A3G, 0.80; +VifHXB2, 0.20; +VifIIIB, 0.76; in C, A3G, 1.20; +VifHXB2, 0.16; +VifIIIB, 0.40.
Mentions: To investigate how VifIIIB and VifHXB2 inhibit A3G-induced mutagenesis and why these variants differ in their effect (Fig. 1 and Tables 1 and 2), we conducted assays measuring A3G-catalyzed deaminations on synthetic DNA substrates. Specifically, the assay can measure the processive scanning behavior of A3G by using a 118-nt ssDNA substrate with two 5′-CCC deamination motifs spaced 61 nt apart (Fig. 3A, schematic). Fig. 3A shows a characteristic result of A3G in this assay. A3G deaminated both the 5′- and 3′-proximal CCC motifs, but there was a 2-fold preference for the 5′-motif due to an inherent catalytic orientation specificity of the active site (22). Of importance is the presence of a double deamination band, which indicates that a deamination occurred at both the 5′-C and 3′-C on the same ssDNA (Fig. 3A, 63 nt). The processivity factor is calculated as a ratio of the intensity of this band to the calculated expected value of deaminations that would occur at both 5′- and 3′-proximal motifs if the enzyme were not processive (see “Experimental Procedures”). The processivity factor of 9.1 (Fig. 3A, below gel) means that A3G is 9 times more likely to undergo a processive deamination of both CCC motifs than to deaminate each of the motifs in separate enzyme-ssDNA encounters. VifHXB2 and VifIIIB were able to decrease the processivity of A3G by at least 1.5-fold from 9.1 to 4.1 or 5.6, respectively (Fig. 3A).

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