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.