Research Group of T. Gramberg

Viruses of the HIV-2/SIVsm lineage encode the accessory protein Vpx. While Vpx is not required for virus replication in activated T-cells, it facilitates the infection of myeloid target cells, like macrophages and dendritic cells. In these cells, Vpx has been shown to neutralize the antiviral activity of the restriction factor SAMHD1. Mutations in SAMHD1 are known to cause the Aicardi-Goutieres syndrome (AGS) in humans. AGS is a hereditary autoimmune disease caused by an immune response to accumulating intracellular nucleic acids. SAMHD1 has been shown to act as a dNTP triphosphohydrolase and it has been suggested that SAMHD1 inhibits retroviral reverse transcription by depleting the intracellular dNTP pool. We were part of a study showing for the first time that SAMHD1 restricts infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs) in primary cells, thereby lowering their concentrations below those required for retroviral DNA synthesis. In addition, we also contributed to the finding that SAMHD1 is not only active in myeloid cells, but also in resting CD4⁺ T-cells, which are known to be highly resistant to productive HIV-1 infection. Furthermore, we found that not only HIV and SIV are blocked by SAMHD1, but many diverse retroviruses belonging to the alpha-, beta-, and gamma-retrovirus genera are affected by the SAMHD1-mediated block to reverse transcription.

Human cytomegalovirus belongs to the herpesvirus family and is of great clinical importance, especially during pregnancy or in immunosuppressed patients such as transplant recipients. Although the antiviral activity of SAMHD1 towards DNA viruses has been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains elusive. To determine the impact of SAMHD1 on CMV replication, we used murine CMV (MCMV) to infect our previously established SAMHD1 KO mouse model and found that SAMHD1 inhibits MCMV replication in vivo. We report that murine CMV has evolved a tool to counteract SAMHD1 and employs a novel mechanism to inactivate the restriction factor. By comparing MCMV replication in vitro in myeloid cells and fibroblasts from SAMHD1 KO and control mice, we found that the viral kinase, M97, actively counteracts SAMHD1 by inducing its phosphorylation at the regulatory residue threonine 603, thereby inactivating SAMHD1. The phosphorylation of SAMHD1 in infected cells correlated with a reduced dNTP hydrolase activity and the loss of viral restriction. In addition, we showed for the first time that SAMHD1 acts as a bona fide restriction factor against a naturally occurring virus in vivo. Using our previously established SAMHD1 KO mouse model, we found that SAMHD1 blocks the in vitro and in vivo replication of murine CMV, the well-established model for the human pathogen HCMV. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and identify the M97-mediated phosphorylation of SAMHD1 as a so far undescribed viral countermeasure. Our findings explain how CMV is able to replicate in its target cells and demonstrate the importance of the inactivation of SAMHD1 by the viral kinase for in vivo replication, which makes the M97-mediated inactivation of SAMHD1 an attractive drug target.

The accumulation of intracellular nucleic acids derived from endogenous retroelements thriving in the absence of SAMHD1 has been discussed as potential trigger of the autoimmune disease AGS. Long interspersed element 1 (LINE-1) is the only autonomously active retrotransposon in humans and about 17% of the genome is derived from LINE1 sequences. Novel LINE-1 retrotransposition events can destabilize genome integrity and cause disease by insertional mutagenesis, insertion of splice sites, recombination, transcriptional activation of nearby genes, or by the activation of non-autonomous short interspersed elements (SINEs), like Alu elements. To this date, novel L1-mediated retrotransposition events have been identified as the disease-causing mutations in more than 120 patients.

We showed in vitro that SAMHD1 indeed inhibits the replication of L1 and other endogenous retroelements in cycling cells. By applying GFP- and neomycin-based reporter assays we found also the anti-L1 activity of SAMHD1 to be regulated by phosphorylation at T592. Similar to the block of HIV, the cofactor binding site and the enzymatic active HD domain of SAMHD1 proofed to be essential for restriction of L1 elements. Interestingly, phosphorylation at T592 did not correlate with the dNTP hydrolase activity of SAMHD1 in cycling 293T cells suggesting an alternative but similar mechanism of regulation. Our results suggest that SAMHD1 is important for maintaining genome integrity and support the idea of an enhanced replication of endogenous retroelements in the absence of SAMHD1 in vivo, potentially triggering autoimmune diseases like AGS.