Degradation of SAMHD1 by Vpx Is Independent of Uncoating

Abstract

Sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm. IMPORTANCE SAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation.Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

“…Whereas reports of Vpr shedding from HIV-1 cores are scarce, ultrastructural, biochemical and functional studies found that HIV-2- and SIV-encoded Vpx is loosely associated with the core and is readily released after virus entry [ 48 – 50 ] (see [ 51 ] for the opposite conclusion). Interestingly, virus-incorporated Vpx is rapidly released from post-fusion cores, accumulates in the nucleus and initiates SAMHD1 degradation in HeLa-derived cells and primary macrophages within a few hours after inoculation [ 49 , 50 ]. It is thus tempting to speculate that nuclear accumulation of HIV-1 Vpr observed in our study may also play a yet unknown role in establishment of productive infection.…”

The Per-1 Short Isoform Inhibits de novo HIV-1 Transcription in Resting CD4+ T-cells

“…The pNL4.3.Luc-Tat null vector was gifted by Dr. Guangxia Gao. The vectors producing VLP-Vpx were gifted by Dr. Landau [52], and 8 µg of SIV3 + was cotransfected with 2 μg of VSV-G-encoding plasmid for VLP-Vpx production. VLP-Vpx treatment was performed for 3 h before infection by adding RPMI-diluted VLP-Vpx to the cells, as described previously [50, 51].…”

An Aicardi-Goutières syndrome associated with a quasi-Moyamoya by a biallelic mutation in SAMHD1

“…D'un point de vue fonctionnel, SAMHD1 participerait à l'homéostasie vasculaire, particulièrement cérébrale 6,16 , à la suppression de phénomènes immuns par le clivage direct d'acides nucléiques viraux ou endogènes ainsi qu'à la restriction de la réplication d'HIV-1 dans certaines lignées cellulaires 20 . À noter aussi, une zone d'interaction avec Vpx, une protéine associée au virion de HIV-2 qui déclencherait la dégradation de SAMHD1 et offrirait une résistance virale à ce moyen de défense 21 . Aux niveaux histologique, cellulaire et moléculaire, sa déficience génétique serait la cause d'une atteinte ar térielle inflammatoire 6,15 , d'une translocation cytosolique avec une tendance à l'accumulation pathologique là où la protéine intègre montre une localisation strictement nucléaire 22 et de multiples délétions d'ADN mitochondrial lui ont été imputées 23 .…”