Differential dysregulation of β-TrCP1 and-2 by HIV-1 Vpu leads to inhibition of canonical and non-canonical NF-κB pathways in infected cells

The NF-& kappa;B pathway regulates host responses to infection and is a common target of viral antagonism. The HIV-1 Vpu protein inhibits NF-& kappa;B signaling late in the virus lifecycle, by binding and inhibiting & beta;-TrCP, the substrate recognition portion of the ubiquitin ligase responsible for inducing I & kappa;B degradation. Here we demonstrate that Vpu simultaneously inhibits and exploits the two different paralogues of & beta;-TrCP by triggering the degradation of & beta;-TrCP1 and co-opting & beta;-TrCP2 for the destruction of its cellular targets. In so doing, it has a potent inhibitory effect on both the canonical and non-canonical NF-& kappa;B pathways. This effect has been underestimated in previous mechanistic studies due to the use of Vpu proteins from lab-adapted viruses. Our findings reveal previously unappreciated differences in the & beta;-TrCP paralogues, revealing functional insights into the regulation of these proteins. This study also raises important implications for the role of NF-& kappa;B inhibition in the immunopathogenesis of HIV/AIDS and the way that this may impact on HIV latency reversal strategies based on the activation of the non-canonical NF-& kappa;B pathway. The HIV-1 Vpu protein is expressed late in the virus lifecycle to promote infectious virus production and avoid innate and adaptive immunity. This includes the inhibition of the NF-& kappa;B pathway which, when activated, leads to the induction of inflammatory responses and the promotion of antiviral immunity. Here we demonstrate that Vpu can inhibit both canonical and non-canonical NF-& kappa;B pathways, through the direct inhibition of the F-box protein & beta;-TrCP, the substrate recognition portion of the Skp1-Cul1-F-box (SCF)(& beta;-TrCP) ubiquitin ligase complex. There are two paralogues of & beta;-TrCP (& beta;-TrCP1/BTRC and & beta;-TrCP2/FBXW11), encoded on different chromosomes, which appear to be functionally redundant. Vpu, however, is one of the few & beta;-TrCP substrates to differentiate between the two paralogues. We have found that patient-derived alleles of Vpu, unlike those from lab-adapted viruses, trigger the degradation of & beta;-TrCP1 while co-opting its paralogue & beta;-TrCP2 for the degradation of cellular targets of Vpu, such as CD4. The potency of this dual inhibition correlates with stabilization of the classical I & kappa;B & alpha; and the phosphorylated precursors of the mature DNA-binding subunits of canonical and non-canonical NF-& kappa;B pathways, p105/NF & kappa;B1 and p100/NF & kappa;B2, in HIV-1 infected CD4(+) T cells. Both precursors act as alternative I & kappa;Bs in their own right, thus reinforcing NF-& kappa;B inhibition at steady state and upon activation with either selective canonical or non-canonical NF-& kappa;B stimuli. These data reveal the complex regulation of NF-& kappa;B late in the viral replication cycle, with consequences for both the pathogenesis of HIV/AIDS and the use of NF-& kappa;B-modulating drugs in HIV cure strategies. IMPORTANCEThe NF-& kappa;B pathway regulates host responses to infection and is a common target of viral antagonism. The HIV-1 Vpu protein inhibits NF-& kappa;B signaling late in the virus lifecycle, by binding and inhibiting & beta;-TrCP, the substrate recognition portion of the ubiquitin ligase responsible for inducing I & kappa;B degradation. Here we demonstrate that Vpu simultaneously inhibits and exploits the two different paralogues of & beta;-TrCP by triggering the degradation of & beta;-TrCP1 and co-opting & beta;-TrCP2 for the destruction of its cellular targets. In so doing, it has a potent inhibitory effect on both the canonical and non-canonical NF-& kappa;B pathways. This effect has been underestimated in previous mechanistic studies due to the use of Vpu proteins from lab-adapted viruses. Our findings reveal previously unappreciated differences in the & beta;-TrCP paralogues, revealing functional insights into the regulation of these proteins. This study also raises important implications for the role of NF-& kappa;B inhibition in the immunopathogenesis of HIV/AIDS and the way that this may impact on HIV latency reversal strategies based on the activation of the non-canonical NF-& kappa;B pathway.

Automated summary

The HIV-1 Vpu protein inhibits NF-κB signaling by binding and inhibiting β-TrCP, and it simultaneously exploits the different paralogues of β-TrCP to degrade its cellular targets, resulting in potent inhibition of both the canonical and non-canonical NF-κB pathways.