Contribution of oligomerization to the anti-HIV-1 properties of SAMHD1

Abstract

BackgroundSAMHD1 is a restriction factor that potently blocks infection by HIV-1 and other retroviruses. We have previously demonstrated that SAMHD1 oligomerizes in mammalian cells by immunoprecipitation. Here we investigated the contribution of SAMHD1 oligomerization to retroviral restriction.ResultsStructural analysis of SAMHD1 and homologous HD domain proteins revealed that key hydrophobic residues Y146, Y154, L428 and Y432 stabilize the extensive dimer interface observed in the SAMHD1 crystal structure. Full-length SAMHD1 variants Y146S/Y154S and L428S/Y432S lost their ability to oligomerize tested by immunoprecipitation in mammalian cells. In agreement with these observations, the Y146S/Y154S variant of a bacterial construct expressing the HD domain of human SAMHD1 (residues 109–626) disrupted the dGTP-dependent tetramerization of SAMHD1 in vitro. Tetramerization-defective variants of the full-length SAMHD1 immunoprecipitated from mammalian cells and of the bacterially-expressed HD domain construct lost their dNTPase activity. The nuclease activity of the HD domain construct was not perturbed by the Y146S/Y154S mutations. Remarkably, oligomerization-deficient SAMHD1 variants potently restricted HIV-1 infection.ConclusionsThese results suggested that SAMHD1 oligomerization is not required for the ability of the protein to block HIV-1 infection.

SAMHD1: At the Crossroads of Cell Proliferation, Immune Responses, and Virus Restriction

“…TREX1 is able to digest mislocalized cytoplasmic DNA (from endogenous retroelements or retroviral DNA intermediates), preventing the activation of DNA sensors such as cGAS. The characterization of SAMHD1 mutations, which induce loss of function through both RNase and dNTPase activities or exclusively by either RNase or dNTPase activity, yielded the hypothesis that RNase function of SAMHD1 is essential for HIV-1 restriction [25,36,42]. Thus, an alternative mode of action to explain SAMHD1-mediated viral restriction has been proposed in which SAMHD1 specifically targets HIV-1 RNA for degradation before it is reverse-transcribed in the cytoplasm ( Figure 1B).…”

“…A study of SAMHD1 mutations that abolish SAMHD1 dNTPase, but not RNAse activity, or prevent protein oligomerization, suggested that, unlike the dNTPase activity, the tetramerization of SAMHD1 is not necessary for its RNase activity [25,36]. Under these circumstances, SAMHD1 can be presented as a dual-function enzyme that has mutually exclusive and predominant activities that depend on the dGTP pool.…”

“…At low dGTP levels, SAMHD1 exists as monomers or dimers that harbor active RNAse but not dNTPase activity, enabling HIV-1 restriction through the cleavage of HIV-1 RNA. At high dGTP levels, SAMHD1 tetramerizes, yielding an enzyme with active dNTPase function [25,36].…”

dNTP pool modulation dynamics by SAMHD1 protein in monocyte-derived macrophages

“…The nuclease activity was localized to the HD domain of SAMHD1 [31]. Both single-stranded DNA and RNA nuclease activities have been reported for SAMHD1 [29,32].…”

“…First, SAMHD1 was shown to have deoxyribonucleoside triphosphate (dNTP) phosphohydrolase activity [20,21], suggesting it is a host antiviral restriction factor to limit replication of retroviral and DNA containing viruses by depleting cellular dNTPs in viral non-dividing target cell types [22][23][24][25]. Recently, both biochemical and structural evidence indicated that SAMHD1 forms a tetramer as the active dNTP phosphohydrolase complex [26][27][28][29]. When dNTPs bind in the active site, the tetramer is formed, and the tetramer was suggested to be long-lived in the cell [30].…”