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Requirement of HIV-1 Vif C-terminus for Vif-CBF-β interaction and assembly of CUL5-containing E3 ligase.

Wang H, Lv G, Zhou X, Li Z, Liu X, Yu XF, Zhang W - BMC Microbiol. (2014)

Bottom Line: Moreover, by determining that the BC box also is necessary for CBF-β interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-β and consequent interaction with CUL5.These results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase.Identification of the new binding interface with CBF-β at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and AIDS Research, First Hospital of Jilin University, No 519. East Minzhu Avenue, Changchun, Jilin Province, China. wanghong309768094@126.com.

ABSTRACT

Background: Human immunodeficiency virus type 1 (HIV-1) Vif hijacks an E3 ligase to suppress natural APOBEC3 restriction factors, and core binding factor β (CBF-β) is required for this process. Although an extensive region of Vif spanning most of its N-terminus is known to be critical for binding with CBF-β, involvement of the Vif C-terminus in the interaction with CBF-β has not been fully investigated.

Results: Here, through immunoprecipitation analysis of Vif C-terminal truncated mutants of various lengths, we identified that CBF-β binding requires not only certain amino acids (G126A, E134A, Y135A and G138A) in the HCCH region but also the HCCH motif itself, which also affects the Vif-mediated suppression of APOBEC3G/APOBEC3F (A3G/A3F). These mutants still maintained interactions with substrate A3G or A3F as well as other cellular factors ElonginB/C (ELOB/C), indicating that their structures were not functionally affected. Moreover, by determining that the BC box also is necessary for CBF-β interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-β and consequent interaction with CUL5.

Conclusions: These results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase. Identification of the new binding interface with CBF-β at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors.

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Effects of Vif mutations in the region from amino acid 124-141 on the antiviral activity of A3G. (A) Effects of WT and mutant Vif proteins on A3G antiviral activity. HIV-1 viruses were produced by transfecting HEK293T cells with NL4-3ΔVif and A3G along with VR1012 as a control vector or WT or various Vif mutants as indicated. Virus infectivity was assessed using MAGI indicator cells, with the virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviation from triplicate wells. (B) Effects of WT and mutant Vif proteins on A3G degradation and virion packaging. A3G expression was assessed by Western blotting against A3G-HA, Vif-HA and tubulin as loading control. A3G packaging was evaluated by Western blotting against A3G-HA and CAp24 after virus was purified from the cell culture supernatant.
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Fig4: Effects of Vif mutations in the region from amino acid 124-141 on the antiviral activity of A3G. (A) Effects of WT and mutant Vif proteins on A3G antiviral activity. HIV-1 viruses were produced by transfecting HEK293T cells with NL4-3ΔVif and A3G along with VR1012 as a control vector or WT or various Vif mutants as indicated. Virus infectivity was assessed using MAGI indicator cells, with the virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviation from triplicate wells. (B) Effects of WT and mutant Vif proteins on A3G degradation and virion packaging. A3G expression was assessed by Western blotting against A3G-HA, Vif-HA and tubulin as loading control. A3G packaging was evaluated by Western blotting against A3G-HA and CAp24 after virus was purified from the cell culture supernatant.

Mentions: After identifying the CBF-β binding residues in the Vif HCCH region, we further analyzed the effect of these Vif mutants on the antiviral activity of A3G proteins. The MAGI assay was used to analyze the function of Vif mutants against A3G (Figure 4). As expected, Vif mutants L125A, G126A, R127A, C133A, E134A, Y135A, G138A and H139A were defective in the suppression of A3G antiviral activity (Figure 4A). As a control, other Vif mutants, including I128A, V129A, S130A, P131A, R132A, Q136A, A137S, N140A and K141A, showed abilities to inhibit A3G comparable to that of WT Vif. To further confirm whether these Vif mutations that abolished the A3G suppression are due to reduced abilities to degrade and exclude A3G from virons, A3G expression levels in cells and virions were analyzed (Figure 4B). The Vif mutants L125A, G126A, R127A, C133A, E134A, Y135A, G138A and H139A (Figure 4B, lanes 3, 4, 5, 13, 14, 17, 20 and 23) were found to be defective in degrading A3G and excluding it from HIV-1 virions compared with WT Vif (Figure 4B, lanes 2, 8, 16 and 22).Figure 4


Requirement of HIV-1 Vif C-terminus for Vif-CBF-β interaction and assembly of CUL5-containing E3 ligase.

Wang H, Lv G, Zhou X, Li Z, Liu X, Yu XF, Zhang W - BMC Microbiol. (2014)

Effects of Vif mutations in the region from amino acid 124-141 on the antiviral activity of A3G. (A) Effects of WT and mutant Vif proteins on A3G antiviral activity. HIV-1 viruses were produced by transfecting HEK293T cells with NL4-3ΔVif and A3G along with VR1012 as a control vector or WT or various Vif mutants as indicated. Virus infectivity was assessed using MAGI indicator cells, with the virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviation from triplicate wells. (B) Effects of WT and mutant Vif proteins on A3G degradation and virion packaging. A3G expression was assessed by Western blotting against A3G-HA, Vif-HA and tubulin as loading control. A3G packaging was evaluated by Western blotting against A3G-HA and CAp24 after virus was purified from the cell culture supernatant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265484&req=5

Fig4: Effects of Vif mutations in the region from amino acid 124-141 on the antiviral activity of A3G. (A) Effects of WT and mutant Vif proteins on A3G antiviral activity. HIV-1 viruses were produced by transfecting HEK293T cells with NL4-3ΔVif and A3G along with VR1012 as a control vector or WT or various Vif mutants as indicated. Virus infectivity was assessed using MAGI indicator cells, with the virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviation from triplicate wells. (B) Effects of WT and mutant Vif proteins on A3G degradation and virion packaging. A3G expression was assessed by Western blotting against A3G-HA, Vif-HA and tubulin as loading control. A3G packaging was evaluated by Western blotting against A3G-HA and CAp24 after virus was purified from the cell culture supernatant.
Mentions: After identifying the CBF-β binding residues in the Vif HCCH region, we further analyzed the effect of these Vif mutants on the antiviral activity of A3G proteins. The MAGI assay was used to analyze the function of Vif mutants against A3G (Figure 4). As expected, Vif mutants L125A, G126A, R127A, C133A, E134A, Y135A, G138A and H139A were defective in the suppression of A3G antiviral activity (Figure 4A). As a control, other Vif mutants, including I128A, V129A, S130A, P131A, R132A, Q136A, A137S, N140A and K141A, showed abilities to inhibit A3G comparable to that of WT Vif. To further confirm whether these Vif mutations that abolished the A3G suppression are due to reduced abilities to degrade and exclude A3G from virons, A3G expression levels in cells and virions were analyzed (Figure 4B). The Vif mutants L125A, G126A, R127A, C133A, E134A, Y135A, G138A and H139A (Figure 4B, lanes 3, 4, 5, 13, 14, 17, 20 and 23) were found to be defective in degrading A3G and excluding it from HIV-1 virions compared with WT Vif (Figure 4B, lanes 2, 8, 16 and 22).Figure 4

Bottom Line: Moreover, by determining that the BC box also is necessary for CBF-β interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-β and consequent interaction with CUL5.These results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase.Identification of the new binding interface with CBF-β at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and AIDS Research, First Hospital of Jilin University, No 519. East Minzhu Avenue, Changchun, Jilin Province, China. wanghong309768094@126.com.

ABSTRACT

Background: Human immunodeficiency virus type 1 (HIV-1) Vif hijacks an E3 ligase to suppress natural APOBEC3 restriction factors, and core binding factor β (CBF-β) is required for this process. Although an extensive region of Vif spanning most of its N-terminus is known to be critical for binding with CBF-β, involvement of the Vif C-terminus in the interaction with CBF-β has not been fully investigated.

Results: Here, through immunoprecipitation analysis of Vif C-terminal truncated mutants of various lengths, we identified that CBF-β binding requires not only certain amino acids (G126A, E134A, Y135A and G138A) in the HCCH region but also the HCCH motif itself, which also affects the Vif-mediated suppression of APOBEC3G/APOBEC3F (A3G/A3F). These mutants still maintained interactions with substrate A3G or A3F as well as other cellular factors ElonginB/C (ELOB/C), indicating that their structures were not functionally affected. Moreover, by determining that the BC box also is necessary for CBF-β interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-β and consequent interaction with CUL5.

Conclusions: These results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase. Identification of the new binding interface with CBF-β at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors.

Show MeSH
Related in: MedlinePlus