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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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Vif-CBFβ-EloB/C is an RNA-independent stable complex in solution.(A) Vif-CBFβ-EloB/C was not dependent on RNA. The purified complex (2 mg/ml, 100 µl) was incubated with 2 µl RNase Mix (RNase A/T1 Mix, Catalog EN055, Fermentas) at 37°C for 4 h according to the manufacturer's protocol, followed by buffer exchange to remove the EDTA. The complex then was analyzed by His-tag affinity pull-down. (B) The Vif-CBFβ complex is not stable. Purified Vif-CBFβ complexes were concentrated to 4 mg/ml and, after clarification at 13,000 g for 10 min, the supernatants were stored at 4°C (Input). Samples were then removed at different times (0 h, 6 h, 24 h), and after clarification at 13,000 g for 10 min, the supernatants (S) were removed and the pellets (P) resuspended to the original volume and checked by SDS-PAGE. (C) Purified Vif complexes were concentrated to 5 mg/ml (Input) and stored at 4°C overnight (∼16 h). The supernatants (S) were removed after clarification at 13,000 g for 10 min, and the pellets (P) were resuspended to the original volume, then checked by SDS-PAGE.
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pone-0033495-g005: Vif-CBFβ-EloB/C is an RNA-independent stable complex in solution.(A) Vif-CBFβ-EloB/C was not dependent on RNA. The purified complex (2 mg/ml, 100 µl) was incubated with 2 µl RNase Mix (RNase A/T1 Mix, Catalog EN055, Fermentas) at 37°C for 4 h according to the manufacturer's protocol, followed by buffer exchange to remove the EDTA. The complex then was analyzed by His-tag affinity pull-down. (B) The Vif-CBFβ complex is not stable. Purified Vif-CBFβ complexes were concentrated to 4 mg/ml and, after clarification at 13,000 g for 10 min, the supernatants were stored at 4°C (Input). Samples were then removed at different times (0 h, 6 h, 24 h), and after clarification at 13,000 g for 10 min, the supernatants (S) were removed and the pellets (P) resuspended to the original volume and checked by SDS-PAGE. (C) Purified Vif complexes were concentrated to 5 mg/ml (Input) and stored at 4°C overnight (∼16 h). The supernatants (S) were removed after clarification at 13,000 g for 10 min, and the pellets (P) were resuspended to the original volume, then checked by SDS-PAGE.

Mentions: The stability of the purified Vif-CBFβ140 complexes was low: at 4°C, the complexes precipitated after only a few hours (Fig. 5B). After 16 h at 4°C, >50% of the Vif protein precipitated (Fig. 5C, lanes 1–3). More Vif protein than CBFβ140 protein appeared in the precipitates, although the initial ratio of Vif and CBFβ was about 1∶1 (Fig. 5C, lane 1).In contrast, the Vif-CBFβ140-EloB/C complexes were more stable (Fig. 5A, lanes 4–6): only a trace amount of Vif precipitated after 16 h at 4°C.


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)

Vif-CBFβ-EloB/C is an RNA-independent stable complex in solution.(A) Vif-CBFβ-EloB/C was not dependent on RNA. The purified complex (2 mg/ml, 100 µl) was incubated with 2 µl RNase Mix (RNase A/T1 Mix, Catalog EN055, Fermentas) at 37°C for 4 h according to the manufacturer's protocol, followed by buffer exchange to remove the EDTA. The complex then was analyzed by His-tag affinity pull-down. (B) The Vif-CBFβ complex is not stable. Purified Vif-CBFβ complexes were concentrated to 4 mg/ml and, after clarification at 13,000 g for 10 min, the supernatants were stored at 4°C (Input). Samples were then removed at different times (0 h, 6 h, 24 h), and after clarification at 13,000 g for 10 min, the supernatants (S) were removed and the pellets (P) resuspended to the original volume and checked by SDS-PAGE. (C) Purified Vif complexes were concentrated to 5 mg/ml (Input) and stored at 4°C overnight (∼16 h). The supernatants (S) were removed after clarification at 13,000 g for 10 min, and the pellets (P) were resuspended to the original volume, then checked by SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033495-g005: Vif-CBFβ-EloB/C is an RNA-independent stable complex in solution.(A) Vif-CBFβ-EloB/C was not dependent on RNA. The purified complex (2 mg/ml, 100 µl) was incubated with 2 µl RNase Mix (RNase A/T1 Mix, Catalog EN055, Fermentas) at 37°C for 4 h according to the manufacturer's protocol, followed by buffer exchange to remove the EDTA. The complex then was analyzed by His-tag affinity pull-down. (B) The Vif-CBFβ complex is not stable. Purified Vif-CBFβ complexes were concentrated to 4 mg/ml and, after clarification at 13,000 g for 10 min, the supernatants were stored at 4°C (Input). Samples were then removed at different times (0 h, 6 h, 24 h), and after clarification at 13,000 g for 10 min, the supernatants (S) were removed and the pellets (P) resuspended to the original volume and checked by SDS-PAGE. (C) Purified Vif complexes were concentrated to 5 mg/ml (Input) and stored at 4°C overnight (∼16 h). The supernatants (S) were removed after clarification at 13,000 g for 10 min, and the pellets (P) were resuspended to the original volume, then checked by SDS-PAGE.
Mentions: The stability of the purified Vif-CBFβ140 complexes was low: at 4°C, the complexes precipitated after only a few hours (Fig. 5B). After 16 h at 4°C, >50% of the Vif protein precipitated (Fig. 5C, lanes 1–3). More Vif protein than CBFβ140 protein appeared in the precipitates, although the initial ratio of Vif and CBFβ was about 1∶1 (Fig. 5C, lane 1).In contrast, the Vif-CBFβ140-EloB/C complexes were more stable (Fig. 5A, lanes 4–6): only a trace amount of Vif precipitated after 16 h at 4°C.

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