Nuclear import of SAMHD1 is mediated by a classical karyopherin α/β1 dependent pathway and confers sensitivity to VpxMAC induced ubiquitination and proteasomal degradation.
BACKGROUND
The deoxynucleotide-triphosphate (dNTP) hydrolase sterile alpha motif domain and HD domain 1 (SAMHD1) is a nuclear protein that inhibits HIV-1 infection in myeloid cells as well as quiescent CD4 T-cells, by decreasing the intracellular dNTP concentration below a level that is required for efficient reverse transcription. The Vpx proteins of the SIVSMM/HIV-2 lineage of lentiviruses bind SAMHD1 and recruit an ubiquitin ligase, leading to polyubiquitination and proteasomal degradation.


RESULTS
Here, we have investigated the importance of nuclear localization for SAMHD1's antiviral function as well as its sensitivity to the Vpx protein of SIVMAC. Using GST pull down assays, as well as RNA silencing approaches, we show that SAMHD1 preferentially uses karyopherin α2 (KPNA2) and a classical N-terminal nuclear localization signal (14KRPR17) to enter the nucleus. Reduction of karyopherin β1 (KPNB1) or KPNA2 by RNAi also led to cytoplasmic re-distribution of SAMHD1. Using primary human monocyte-derived macrophages (MDM), a cell type in which SAMHD1 is naturally expressed to high levels, we demonstrate that nuclear localization is not required for its antiviral activity. Cytoplasmic SAMHD1 still binds to VpxMAC, is efficiently polyubiquitinated, but is not degraded. We also find that VpxMAC-induced SAMHD1 degradation was partially reversed by ubiquitin carrying the K48R or K11R substitution mutations, suggesting involvement of K48 and K11 linkages in SAMHD1 polyubiquitination. Using ubiquitin K-R mutants also revealed differences in the ubiquitin linkages between wild type and cytoplasmic forms of SAMHD1, suggesting a potential association with the resistance of cytoplasmic SAMHD1 to VpxMAC induced degradation.


CONCLUSIONS
Our work extends published observations on SAMHD1 nuclear localization to a natural cell type for HIV-1 infection, identifies KPNA2/KPNB1 as cellular proteins important for SAMHD1 nuclear import, and indicates that components of the nuclear proteasomal degradation machinery are required for SAMHD1 degradation.
Department of Infectious Diseases, King's College London, 2nd Floor, Borough Wing, Guy's Hospital, London Bridge, London SE1 9RT, UK. torsten.schaller@kcl.ac.uk.
Torsten
T
Schaller
Darja
D
Pollpeter
Luis
L
Apolonia
Caroline
C
Goujon
Michael H
MH
Malim
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D028884:Karyopherins / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000821:virology; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis; D000076106:SAM Domain and HD Domain-Containing Protein 1; D054875:Ubiquitination; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D028884:Karyopherins; C513059:VPX protein, Simian immunodeficiency virus; D054334:Viral Regulatory and Accessory Proteins; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human; D046988:Proteasome Endopeptidase Complex; D020559:Monomeric GTP-Binding Proteins