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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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Mutation cluster frequency of A3G is decreased in the presence of VifIIIB but not VifHXB2. Sliding movement of A3G is inferred from clustered mutations. The frequency of clustered mutations was scored in eight regions of the prot-lacZα that contain three to six G residues. Horizontal bars represent the average mutation cluster frequency. The data show that A3G alone and in the presence of VifHXB2 induced a similar frequency of clustered mutations (p value, 0.32). However, A3G in the presence of VifIIIB induced significantly fewer clustered mutations (p value, 0.02).
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Figure 4: Mutation cluster frequency of A3G is decreased in the presence of VifIIIB but not VifHXB2. Sliding movement of A3G is inferred from clustered mutations. The frequency of clustered mutations was scored in eight regions of the prot-lacZα that contain three to six G residues. Horizontal bars represent the average mutation cluster frequency. The data show that A3G alone and in the presence of VifHXB2 induced a similar frequency of clustered mutations (p value, 0.32). However, A3G in the presence of VifIIIB induced significantly fewer clustered mutations (p value, 0.02).

Mentions: To further test this hypothesis, we examined the sequences of clones from the HIV-1 replication assay. We can analyze the spatial proximity of mutations in the clones and relate it to processivity where clustered mutations are indicative of scanning by local sliding and distantly spaced singleton mutations are indicative of scanning by jumping (19). Areas of the 368-nt (+)DNA sequence (containing prot and lacZα), which contains multiple G residues, were used to analyze the frequency of clustered A3G-induced G→A mutations. If the sliding component of A3G ssDNA scanning mechanism is retained, A3G should be able to induce mutagenesis at multiple G residues in a close region, i.e. 10 nt or less. The mean clustered mutation frequency for A3G alone (0.48; Fig. 4) is similar to that for the situation when VifHXB2 was present (0.33; p value, 0.32; Fig. 4). These data are consistent with Fig. 3, A–C, which shows that A3G retained the ability to slide, but not jump, in the presence of VifHXB2. In the presence of VifIIIB, A3G was less capable of inducing mutations that are closely spaced (0.14; p value, 0.02; Fig. 4). This indicates that A3G was less able to scan ssDNA by sliding when VifIIIB was present and is in agreement with processivity data obtained from synthetic substrates (Fig. 3, A–C).


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)

Mutation cluster frequency of A3G is decreased in the presence of VifIIIB but not VifHXB2. Sliding movement of A3G is inferred from clustered mutations. The frequency of clustered mutations was scored in eight regions of the prot-lacZα that contain three to six G residues. Horizontal bars represent the average mutation cluster frequency. The data show that A3G alone and in the presence of VifHXB2 induced a similar frequency of clustered mutations (p value, 0.32). However, A3G in the presence of VifIIIB induced significantly fewer clustered mutations (p value, 0.02).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Mutation cluster frequency of A3G is decreased in the presence of VifIIIB but not VifHXB2. Sliding movement of A3G is inferred from clustered mutations. The frequency of clustered mutations was scored in eight regions of the prot-lacZα that contain three to six G residues. Horizontal bars represent the average mutation cluster frequency. The data show that A3G alone and in the presence of VifHXB2 induced a similar frequency of clustered mutations (p value, 0.32). However, A3G in the presence of VifIIIB induced significantly fewer clustered mutations (p value, 0.02).
Mentions: To further test this hypothesis, we examined the sequences of clones from the HIV-1 replication assay. We can analyze the spatial proximity of mutations in the clones and relate it to processivity where clustered mutations are indicative of scanning by local sliding and distantly spaced singleton mutations are indicative of scanning by jumping (19). Areas of the 368-nt (+)DNA sequence (containing prot and lacZα), which contains multiple G residues, were used to analyze the frequency of clustered A3G-induced G→A mutations. If the sliding component of A3G ssDNA scanning mechanism is retained, A3G should be able to induce mutagenesis at multiple G residues in a close region, i.e. 10 nt or less. The mean clustered mutation frequency for A3G alone (0.48; Fig. 4) is similar to that for the situation when VifHXB2 was present (0.33; p value, 0.32; Fig. 4). These data are consistent with Fig. 3, A–C, which shows that A3G retained the ability to slide, but not jump, in the presence of VifHXB2. In the presence of VifIIIB, A3G was less capable of inducing mutations that are closely spaced (0.14; p value, 0.02; Fig. 4). This indicates that A3G was less able to scan ssDNA by sliding when VifIIIB was present and is in agreement with processivity data obtained from synthetic substrates (Fig. 3, A–C).

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