Abstract
BackgroundHijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. In particular, HIV-1 Vpu usurps the SCFβ-TrCP E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. Vpu also interacts with and downmodulates a number of other host proteins, including the restriction factor BST-2. However, whether Vpu primarily relies on a cullin-dependent or -independent mechanism to antagonize its cellular targets has not been fully elucidated.ResultsWe utilized a sulphamate AMP analog, MLN4924, to effectively block the activation of CRLs within infected primary CD4+ T cells. MLN4924 treatment, in a dose dependent manner, efficiently relieved surface downmodulation and degradation of CD4 by NL4-3 Vpu. MLN4924 inhibition was highly specific, as this inhibitor had no effect on Nef’s ability to downregulate CD4, which is accomplished by a CRL-independent mechanism. In contrast, NL4-3 Vpu’s capacity to downregulate BST-2, NTB-A and CCR7 was not inhibited by the drug. Vpu’s from both a transmitted founder (T/F) and chronic carrier (CC) virus preserved the ability to downregulate BST-2 in the presence of MLN4924. Finally, depletion of cellular pools of cullin 1 attenuated Vpu’s ability to decrease CD4 but not BST-2 surface levels.ConclusionsWe conclude that Vpu employs both CRL-dependent and CRL-independent modes of action against host proteins. Notably, we also establish that Vpu-mediated reduction of BST-2 from the cell surface is independent of β-TrCP and the CRL- machinery and this function is conserved by Vpu’s from primary isolates. Therefore, potential therapies aimed at antagonizing the activities of Vpu may need to address these distinct mechanisms of action in order to achieve a maximal effect.
Highlights
Hijacking of the cullin-really interesting new gene (RING) E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins
We establish that viral protein u (Vpu)-mediated reduction of BST-2 from the cell surface is independent of β-TrCP and the cullin-RING E3 ubiquitin ligase (CRL)- machinery and this function is conserved by Vpu’s from primary isolates
Pharmacological inhibition of CRL‐activity disables NL4‐3 Vpu’s ability to downregulate CD4, but not BST‐2, CCR7 or NTB‐A To determine whether Vpu can act as multifunctional protein capable of downregulating host proteins in the absence of neddylation and a functional SCFβ-TrCP complex, primary CD4+ T cells were infected with either an HIV-1NL4-3-derived, replication-defective virus carrying GFP in place of Nef (DHIVGFP(Vpu+/Nef−); Fig. 1a), or with an isogenic virus lacking both Nef and Vpu (DHIVGFP(Vpu−/Nef−); Fig. 1b) [21]
Summary
Hijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. HIV-1 Vpu usurps the SCFβ-TrCP E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. The HIV-1 accessory protein Vif hijacks a cullin-5 containing ubiquitin ligase complex (CRL5) to target cytidine deaminases of the APOBEC3 family for proteasomal degradation [2,3,4,5]. Activation of CRLs is dependent on a process known as neddylation. This post-translational modification involves the covalent addition of the NEDD8 protein, a relative of ubiquitin, onto a lysine residue on the cullin backbone.
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