Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein

Abstract

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.

Figure 1. Vpx-mediated relief of the inhibition of HIV-1 infection in MDM requires Vpx glutamine Q76

a. Alleviation of the inhibition of HIV-1 infection in MDM requires Vpx glutamine Q76. MDM were infected with single cycle HIV-1–GFP reporter virus alone, or co infected with HIV-1–GFP and SIV VLP loaded with wild type or Q76A substituted HIV-2 Rod Vpx. Percent fractions of GFP-positive cells are indicated. b. VpxQ76A does not rescue HIV-1 cDNA synthesis. Quantification of HIV-1 “early” (light bars) and “late” (black bars) cDNA products in MDM co-infected with HIV-1–GFP and SIV VLP loaded with HIV-2 wild type Vpx or Vpx_Q76A. (Boil): boiled HIV-1/SIV VLP control. c. SIV VLP contain similar amounts of wild type and Q76A substituted Vpx. Two-fold dilutions of SIV VLP(Vpx), SIV VLP(Vpx_Q76A) or SIV VLP with no Vpx (none) were immunoblotted for Vpx and p27 capsid.

Figure 2. Vpx recruits SAMHD1 to DDB1-DCAF1 module of CRL4^DCAF1 E3 complex

a. Proteins and complexes purified for MudPIT analyses. Architecture of the quaternary Cullin4-DDB1(DDA1)-DCAF1 complex alone, and with bound Vpx, and the placements of HA- (brown rectangle) and FLAG- (yellow triangle) epitope tags is shown. DDA1 is a DDB1-associated protein that co-purifies with DCAF1-DDB1 sub-complex^,. b. HIV-2 and SIVmac Vpx bind SAMHD1. The indicated FLAG-tagged Vpx and Vpr proteins were transiently co-expressed with myc-tagged SAMHD1 in HEK 293T cells. Cell extracts and α-FLAG immune complexes were immunoblotted for SAMHD1 and Vpx/Vpr. c. Vpx recruits SAMHD1 to DCAF1-DDB1 complex. FLAG-SAMHD1 was transiently co-expressed with myc-tagged SIVmac Vpx, Vpx_Q76A, or HIV-1 Vpr, in HEK293T cells. α-FLAG-SAMHD1 immune complexes were immunoblotted for endogenous DCAF1, DDB1, and ectopically expressed SAMHD1 and Vpr/Vpx.

Figure 3. Vpx programs SAMHD1 for proteasomal degradation in MDM, via CRL4^DCAF1 E3

a. SAMHD1 levels in MDM infected with 3-fold serial dilutions of SIV VLP loaded with wild type or Q76A substituted HIV-2 Rod Vpx, or in mock infected MDM (M). α-tubulin served as loading control. b. SAMHD1 levels in MDM infected with 3-fold dilutions of SIVmac Vpx-loaded SIV VLP and cultured in the presence or absence of MG132 proteasome inhibitor (1 μg/ml). c. Vpx depletes SAMHD1 levels in MDM via DCAF1. MDM were subjected to RNAi targeting DCAF1, SAMHD1 or non-targeting RNAi (NT1, NT2), and infected (+) or not (-) with SIV VLP loaded with SIVmac Vpx, two days later. DCAF1 and SAMHD1 were revealed by Western blotting after additional two days.

Figure 4. SAMHD1 inhibits HIV-1 infection in macrophages

a. RNAi mediated SAMHD1 depletion relieves inhibition of HIV-1 infection in MDM. CD14⁺ monocytes isolated from three donors were infected with decreasing doses of Vpx-loaded SIV VLP, differentiated into MDM, subjected to RNAi targeting SAMHD1 (open circles), or non-targeting RNAi (filled circles), and infected with HIV-1–GFP reporter virus. GFP⁺ MDM were quantified 3 days later (upper panels). Fold-stimulation (Fold x) of HIV-1–GFP transduction of MDM, following RNAi targeting SAMHD1, compared to non-targeting RNAi, is also shown (bottom panels). b. SAMHD1 and α-tubulin control levels in MDM from a typical experiment shown in panel (a). N – non-targeting RNAi, S – RNAi to SAMHD1. c. SAMHD1 depletion relieves HIV-1 cDNA synthesis. “early” (E) and “late” (L) viral cDNA products in MDM exposed to low doses of Vpx-loaded SIV VLP and transfected with siRNA to SAMHD1 (S) or non-targeting siRNA (N), and then challenged with HIV-1–GFP. (Boil): boiled HIV-1/SIV VLP control. d. SAMHD1 inhibits MDM transduction by vpx-defective HIV-2_vpx-–GFP and SIVmac_vpx-–GFP single cycle reporter viruses.