CBFβ Enhances De Novo Protein Biosynthesis of Its Binding Partners HIV-1 Vif and RUNX1 and Potentiates the Vif-Induced Degradation of APOBEC3G

Abstract

Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBF␤ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBF␤ remains unclear. Several studies noted reduced Vif expression in CBF␤ knockdown cells while others saw no significant impact of CBF␤ on Vif stability. Here, we confirmed that CBF␤ increases Vif steady-state levels. CBF␤ affected expression of neither viral Gag nor Vpu protein, indicating that CBF␤ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBF␤ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBF␤ to interact with Vif. Importantly, at comparable Vif levels, CBF␤ further enhanced A3G degradation, suggesting that CBF␤ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBF␤ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBF␤ had no detectable effect on RUNX1 metabolic stability. We propose that CBF␤ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. IMPORTANCEIn this study, we show that CBF␤ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBF␤. Kinetic studies revealed that CBF␤ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBF␤ were nonresponsive to CBF␤. Our data suggest that CBF␤ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.

Core-Binding Factor Subunit Beta Is Not Required for Non-Primate Lentiviral Vif-Mediated APOBEC3 Degradation

“…A previous study proposed that CBF-␤ promotes steady-state levels of HIV-1 Vif through the effect on HIV-1 Vif metabolism and biosynthesis (38). As mentioned above, we noticed that the steady-state level of HIV-1 Vif was sensitive to CBF-␤ depletion (Fig.…”

“…Recent studies have shown that the transcription cofactor core-binding factor subunit beta (CBF-␤) is a key factor in Vif function (29,30). CBF-␤ interacts with HIV-1 Vif to enhance Vif's biosynthesis and facilitate Vif folding and stability, thereby enhancing the nucleation of the E3 ubiquitin ligase complex, promoting A3G degradation, and supporting viral infectivity (31)(32)(33)(34)(35)(36)(37)(38).…”

Making Sense of Multifunctional Proteins: Human Immunodeficiency Virus Type 1 Accessory and Regulatory Proteins and Connections to Transcription

“…The recruitment of CBFβ to the Vif E3 ligase is surprising given that CBFβ is a transcription cofactor and not a component of a known cellular E3 ligase. Recruitment of CBFβ serves to stabilize HIV-1 Vif and is required for HIV-1 Vif-mediated A3 degradation activity in vivo (Figure 1) (50, 51, 53–55). More recent work has shown that CBFβ is required only for primate lentiviral infection, as it is dispensable for nonprimate Vif function (53, 56).…”

Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity

“…CBFβ normally forms a heterodimer with RUNX family of transcription factors, serving to both stabilize RUNX steady-state levels and to enhance DNA-binding affinity (Huang et al, 2001; Tahirov et al, 2001). Recruitment of CBFβ serves to stabilize HIV-1 Vif and is required for HIV-1 Vif A3 degradation activity in vivo (Hultquist et al, 2012; Jäger et al, 2012b; Kim et al, 2013; Miyagi et al, 2014; Zhang et al, 2012). Recent work has shown that this recruitment alters endogenous RUNX activity through competitive binding of HIV-1 Vif to CBFβ, potentially to the benefit of the virus (Kim et al, 2013; Klase et al, 2014).…”