Nuclear import of SAMHD1 is mediated by a classical karyopherin alpha/beta 1 dependent pathway and confers sensitivity to Vpx(MAC) induced ubiquitination and proteasomal degradation

TY - JOUR

T1 - Nuclear import of SAMHD1 is mediated by a classical karyopherin alpha/beta 1 dependent pathway and confers sensitivity to Vpx(MAC) induced ubiquitination and proteasomal degradation

AU - Schaller, Torsten

AU - Pollpeter, Darja

AU - Apolonia, Luis

AU - Goujon, Caroline

AU - Malim, Michael H.

PY - 2014/4/8

Y1 - 2014/4/8

N2 - 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 alpha 2 (KPNA2) and a classical N-terminal nuclear localization signal ((KRPR17)-K-14) to enter the nucleus. Reduction of karyopherin beta 1 (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 Vpx(MAC), is efficiently polyubiquitinated, but is not degraded. We also find that Vpx(MAC)-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 Vpx(MAC) 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.

AB - 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 alpha 2 (KPNA2) and a classical N-terminal nuclear localization signal ((KRPR17)-K-14) to enter the nucleus. Reduction of karyopherin beta 1 (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 Vpx(MAC), is efficiently polyubiquitinated, but is not degraded. We also find that Vpx(MAC)-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 Vpx(MAC) 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.

KW - SAMHD1

KW - Nuclear import

KW - Karyopherin

KW - Vpx

KW - Macrophages

KW - HIV-1

KW - NLS

KW - Ubiquitin linkage

KW - Innate immunity

KW - KPNA

KW - KPNB

KW - Imp alpha

KW - Imp beta

KW - RESTRICTION FACTOR SAMHD1

KW - CD4(+) T-CELLS

KW - HIV-1 INFECTION

KW - LOCALIZATION SIGNALS

KW - DENDRITIC CELLS

KW - PROTEIN IMPORT

KW - VPX

KW - ALPHA

KW - OCT4

KW - IDENTIFICATION

U2 - 10.1186/1742-4690-11-29

DO - 10.1186/1742-4690-11-29

M3 - Article

SN - 1742-4690

VL - 11

JO - Retrovirology

JF - Retrovirology

M1 - 29

ER -