Inhibition of CUL4A Neddylation Causes a Reversible Block to SAMHD1-Mediated Restriction of HIV-1

Abstract

The deoxynucleoside triphosphohydrolase SAMHD1 restricts retroviral replication in myeloid cells. Human immunodeficiency virus type 2 (HIV-2) and a simian immunodeficiency virus from rhesus macaques (SIVmac) encode Vpx, a virion-packaged accessory protein that counteracts SAMHD1 by inducing its degradation. SAMHD1 is thought to work by depleting the pool of intracellular deoxynucleoside triphosphates but has also been reported to have exonuclease activity that could allow it to degrade the viral genomic RNA or viral reverse-transcribed DNA. To induce the degradation of SAMHD1, Vpx co-opts the cullin4a-based E3 ubiquitin ligase, CRL4. E3 ubiquitin ligases are regulated by the covalent attachment of the ubiquitin-like protein Nedd8 to the cullin subunit. Neddylation can be prevented by MLN4924, a drug that inhibits the nedd8-activating enzyme. We report that MLN4924 inhibits the neddylation of CRL4, blocking Vpx-induced degradation of SAMHD1 and maintaining the restriction. Removal of the drug several hours postinfection released the block. Similarly, Vpx-containing virus-like particles and deoxynucleosides added to the cells more than 24 h postinfection released the SAMHD1-mediated block. Taken together, these findings support deoxynucleoside triphosphate pool depletion as the primary mechanism of SAMHD1 restriction and argue against a nucleolytic mechanism, which would not be reversible. Mammalian cells express antiviral proteins that restrict the replication of viruses like HIV-1 and other lentiviruses. One such restriction factor is SAMHD1, a deoxynucleoside triphosphohydrolase that blocks retrovirus infection at reverse transcription in nondividing myeloid cells, such as macrophages and dendritic cells (1-3). SAMHD1 is also expressed in T cells, where it blocks the infection of resting T cells but has little effect on activated T cells (4, 5). It is thought to work by depleting the pool of intracellular deoxynucleoside (dN) triphosphates (dNTPs) to a level below that which supports reverse transcription (1-3, 6, 7), although other mechanisms have been proposed (8, 9). Viruses have evolved various means of counteracting antiviral host proteins, most notably by encoding accessory proteins (reviewed in reference 10). Lentiviruses, such as human immunodeficiency virus type 2 (HIV-2) and a simian immunodeficiency virus from rhesus macaques (SIVmac), encode Vpx, a virion-packaged accessory protein that is released into the target cell postentry (11). Upon its release, Vpx associates with the E3 ubiquitin ligase CRL4 and recruits SAMHD1 to the complex, inducing its proteasomal degradation (12-14). The degradation of SAMHD1 and subsequent rise in dNTP levels occur within 8 h postinfection, after which reverse transcription resumes (15). SAMHD1 localizes to the nucleus of the cell by an amino-terminal nuclear localization sequence (16,17). Deletion of the nuclear localization sequence relocalizes SAMHD1 to the cytoplasm and causes it to be resistant to Vpx-induced degradation. The treatment of cells with leptomycin B,...

Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly

“…Recently, we and other groups discovered that posttranslational modification by neddylation is critical for Vpx-CRL4 (DCAF1) E3 ligase function (59)(60)(61). A neddylationactivating enzyme (NAE) inhibitor, MLN4924, inhibited the neddylation of Vpx-bound Cullin4 and blocked the Vpx-induced degradation of SAMHD1 proteins (60).…”

“…We and other groups have recently demonstrated that the posttranslational modification process of neddylation, which is a determinant for the activation of Cullin-RING E3 ligases, is essential for the activity of the Vpx-CRL4 (DCAF1) E3 ligase (59)(60)(61). The neddylation inhibitor MLN4924 blocks Vpx function and preserves the antiviral activity of SAMHD1.…”

Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

“…We have previously shown this higher-molecularweight band to be the neddylated form of CUL4 (91). Neddylation is required to activate cullin ubiquitin ligases and is also necessary for Vpr and Vpx functions through CRL4 (91,94,95). To test whether the single, slower-migrating band detected in the nucleus is indeed neddylated CUL4, we treated HEK293T cells with the neddylation inhibitor MLN4924 before subcellular fractionation.…”

Degradation of SAMHD1 by Vpx Is Independent of Uncoating

“…To test whether the degradation of the NLS.GFP.SAM595 reporter protein utilized the same pathway as the full-length SAMHD1, we tested its response to MG132, a proteasome inhibitor, and MLN4924, a neddylation inhibitor, both of which are known to inhibit Vpx function (1,2,19). We found that MG132 prevented degradation of the fusion protein and that MLN4924 partially blocked the degradation (Fig.…”

“…The activity of the CRL4 DCAF1 E3 ubiquitin ligase is regulated by the conjugation of Nedd8 to Cul4A. Inhibition of neddylation using the drug MLN4924 prevents SAMHD1 degradation (19). DCAF1 also functions in HECT-family EDD/UBR5 E3 ubiquitin ligases (re-viewed in reference 20), and HIV-1 Vpr interacts with the EDD-DDB1-DCAF1 E3 ligase complex to inhibit telomerase activity (21).…”