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Characterization of the interaction of full-length HIV-1 Vif protein with its key regulator CBFβ and CRL5 E3 ubiquitin ligase components.

Zhou X, Evans SL, Han X, Liu Y, Yu XF - PLoS ONE (2012)

Bottom Line: Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif.Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5).This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and AIDS Research, First Affiliated Hospital of Jilin University, Jilin, People's Republic of China.

ABSTRACT
Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

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Constructs used in this study.The vectors and their antibiotic resistance are given, followed by a diagram of the construct: Amp+, ampicillin; Kana+, kanamycin; and Cl+, chloramphenicol. Elongins B and C (EloB/C) are in a single vector (pACYC-Duet) regulated by dual promoters. The Precission protease sequence site in glutathione S-transferase (GST) and Cul5 are indicated.
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pone-0033495-g001: Constructs used in this study.The vectors and their antibiotic resistance are given, followed by a diagram of the construct: Amp+, ampicillin; Kana+, kanamycin; and Cl+, chloramphenicol. Elongins B and C (EloB/C) are in a single vector (pACYC-Duet) regulated by dual promoters. The Precission protease sequence site in glutathione S-transferase (GST) and Cul5 are indicated.

Mentions: Full-length Vif192 in the pET21 vector was a gift from Drs. Rahul M. Kohli and James T. Stivers. Truncated Vif176 and Vif140 were cloned into pET21 vector. Elongin B and Elongin C (residues 17 to 112) in the pACYC-Duet plasmid were a gift from Alex Bullock (University of Oxford, Oxford, United Kingdom). Mouse CBFβ (residues 1–187) cDNA were a gift from Nancy A. Speck (University of Pennsylvania). CBFβ isoform 1 (residues 1–187) from mouse, CBFβ isoform 2 (residues 1–182) and truncated CBFβ (residues 1–140) from human were cloned into pRSF-Duet. For expression, the plasmids were transformed into Escherichia coli BL21(DE3) cells. The constructs used in this study are summarized in Fig. 1. The proteins were over-expressed overnight at 16°C by induction with 0.1 mM isopropyl-D-thiogalactopyranoside (IPTG). Harvested cells were lysed in 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl and then clarified by sonication and centrifugation at 13,000 g for 30 min. For solubility analysis, the supernatant was removed and the pellet resuspended to the original volume. For nickel affinity purification, the supernatant was transferred to Ni-NTA beads (Invitrogen), and the flowthrough was loaded onto Ni-NTA beads for two more passages. After washing with 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl and 40 mM imidazole, the protein complex was eluted with 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl and 400 mM imidazole. Gel filtration and anion exchange were utilized to remove trace contamination. Cul5-NTD (residues 1 to 393 with two point mutations, V341R and L345D) in the pGEX-6p-1 vector was expressed in E. coli BL21(DE3) cells overnight at 16°C by induction with 0.1 mM IPTG. Harvested cells were lysed by sonication in 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl, then clarified by centrifugation at 13,000 g for 30 min. The supernatant was transferred to glutathione-Sepharose 4B beads (GE Healthcare) for glutathione S-transferase (GST) affinity chromatography. The GST tag was then removed using Prescission protease. Gel filtration chromatography was utilized for further purification.


Characterization of the interaction of full-length HIV-1 Vif protein with its key regulator CBFβ and CRL5 E3 ubiquitin ligase components.

Zhou X, Evans SL, Han X, Liu Y, Yu XF - PLoS ONE (2012)

Constructs used in this study.The vectors and their antibiotic resistance are given, followed by a diagram of the construct: Amp+, ampicillin; Kana+, kanamycin; and Cl+, chloramphenicol. Elongins B and C (EloB/C) are in a single vector (pACYC-Duet) regulated by dual promoters. The Precission protease sequence site in glutathione S-transferase (GST) and Cul5 are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316577&req=5

pone-0033495-g001: Constructs used in this study.The vectors and their antibiotic resistance are given, followed by a diagram of the construct: Amp+, ampicillin; Kana+, kanamycin; and Cl+, chloramphenicol. Elongins B and C (EloB/C) are in a single vector (pACYC-Duet) regulated by dual promoters. The Precission protease sequence site in glutathione S-transferase (GST) and Cul5 are indicated.
Mentions: Full-length Vif192 in the pET21 vector was a gift from Drs. Rahul M. Kohli and James T. Stivers. Truncated Vif176 and Vif140 were cloned into pET21 vector. Elongin B and Elongin C (residues 17 to 112) in the pACYC-Duet plasmid were a gift from Alex Bullock (University of Oxford, Oxford, United Kingdom). Mouse CBFβ (residues 1–187) cDNA were a gift from Nancy A. Speck (University of Pennsylvania). CBFβ isoform 1 (residues 1–187) from mouse, CBFβ isoform 2 (residues 1–182) and truncated CBFβ (residues 1–140) from human were cloned into pRSF-Duet. For expression, the plasmids were transformed into Escherichia coli BL21(DE3) cells. The constructs used in this study are summarized in Fig. 1. The proteins were over-expressed overnight at 16°C by induction with 0.1 mM isopropyl-D-thiogalactopyranoside (IPTG). Harvested cells were lysed in 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl and then clarified by sonication and centrifugation at 13,000 g for 30 min. For solubility analysis, the supernatant was removed and the pellet resuspended to the original volume. For nickel affinity purification, the supernatant was transferred to Ni-NTA beads (Invitrogen), and the flowthrough was loaded onto Ni-NTA beads for two more passages. After washing with 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl and 40 mM imidazole, the protein complex was eluted with 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl and 400 mM imidazole. Gel filtration and anion exchange were utilized to remove trace contamination. Cul5-NTD (residues 1 to 393 with two point mutations, V341R and L345D) in the pGEX-6p-1 vector was expressed in E. coli BL21(DE3) cells overnight at 16°C by induction with 0.1 mM IPTG. Harvested cells were lysed by sonication in 20 mM Tris-HCl, pH 8.0, with 150 mM NaCl, then clarified by centrifugation at 13,000 g for 30 min. The supernatant was transferred to glutathione-Sepharose 4B beads (GE Healthcare) for glutathione S-transferase (GST) affinity chromatography. The GST tag was then removed using Prescission protease. Gel filtration chromatography was utilized for further purification.

Bottom Line: Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif.Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5).This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and AIDS Research, First Affiliated Hospital of Jilin University, Jilin, People's Republic of China.

ABSTRACT
Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

Show MeSH
Related in: MedlinePlus