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Abstract

Human immunodeficiency virus-1 (HIV-1) and all other viruses are known to interact with multiple host cellular proteins during their replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to repress viral replication. These host cellular proteins are known as restriction factors and they represent the host's first line of defense against the viral pathogens. Sterile alpha motif and HD domain containing 1 (SAMHD1) has been identified as a HIV-1 restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells. SAMHD1 is the target of the viral protein x (Vpx) from simian lentiviruses and HIV-2. Vpx mediates the recruitment of the Cullin4-DDB1-DCAF1 ubiquitin ligase machinery to SAMHD1 leading to polyubiquitination and subsequent degradation of SAMHD1. Previous studies on monocyte-derived dendritic cells suggested that the Vpx-induced rescue of HIV-1 infection from early type I IFN-induced blocks was independent of SAMHD1, since Vpx mutant Q76A, which is unable to recruit DCAF1 and to degrade SAMHD1, still increased HIV-1 infection in type I IFN treated cells. The rescue in healthy blood donor cells was not observed when Q76A mutant Vpx virus-like particles were used, suggesting that – in conflict with previous reports – SAMHD1 degradation is required for efficient Vpx-mediated rescue of HIV-1 from the type I IFN-induced early antiviral blocks. To investigate the role of SAMHD1 in the Vpx-mediated rescue of HIV-1 from the type I IFN-induced block in myeloid cells at more detail, we generated CRISPR/Cas9 THP-1 cells, a monocytic acute myeloid leukemia cell line, lacking a functional SAMHD1 gene. In line with previous studies, the lack of SAMHD1 protein had no impact on the level of the type I IFN-induced early block to HIV-1 infection as compared to control or parental THP-1 cells. However, while Vpx was able to rescue HIV-1 infectivity in parental THP-1 or CRISPR/Cas9 control cells from the type I IFN effects, no rescue was observed when SAMHD1 protein was absent. To investigate whether the enzymatic activity of SAMHD1 was required for the Vpx-mediated rescue of HIV-1 infection from the early type I IFN-induced blocks, we reconstituted expression of wild type or different catalytically-inactive SAMHD1 mutants in SAMHD1-/- cells and found that Vpx increased HIV-1 infectivity in the presence of wild type, but not H233A mutant SAMHD1, suggesting that the enzymatic activity of SAMHD1 is required for a Vpx-induced rescue of HIV-1 infection from the type I IFN-induced block. We also generated a CRISPR/Cas9 THP-1 cell clone, which had one disrupted SAMHD1 allele and one allele, in which the entire nuclear localization signal (11KRPR14) was deleted in frame, generating an internally NLS-disrupted endogenously expressed SAMHD1 protein. In these cells, SAMHD1 was predominantly localized to the cytoplasm, although a 5 fraction was also observed in the nucleoplasm, suggesting for an alternative nuclear import pathway, independent of the classical 11KRPR14 NLS. In these cells, Vpx still rescued HIV-1 from the type I IFN-induced early block to infection. Of note, SAMHD1 degradation was profoundly delayed, suggesting that Vpx-induced polyubiquitination of SAMHD1 is sufficient to overcome the early IFN-induced block to HIV-1 in myeloid cells. SAMHD1 not only acts as a host restriction factor against lentiviral, endogenous retroviruses, hepatitis B virus, herpesviruses (HSV-1) and poxviruses, mutations in the SAMHD1 gene have also been linked to the immune disorder Aicardi-Goutières Syndrome (AGS), a genetic disease mimicking congenital virus infection. Recurrent mutations and reduced expression levels of SAMHD1 have been suggested to play a role for the oncogenesis of several cancers such as colon and Rectum Adenocarcinoma (COAD/READ), lung cancer, cutaneous T-cell lymphoma, acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL). Interestingly, SAMHD1’s function as a possible tumor suppressor is complexed by its role as a resistance factor in nucleoside analogue-based anti-cancer therapies. Cytarabine (ara-C), a deoxycytidine analog, is the single most important drug in the treatment of AML and other hematological malignancies, exerting its cytotoxic effects through its activated triphosphate (ara-CTP), eventually leading to DNA damage and cell death. We and others demonstrated that SAMHD1 is able to detoxify cells by hydrolytic activity towards ara-CTP. Accordingly, primary AML blasts treated with Vpx to deplete SAMHD1 as well as THP-1 CRISPR/Cas9 SAMHD1 knock-out cells showed increased sensitivity towards ara-C induced cytotoxicity. Using these knock-out cells as a back-bone, we expressed a large panel of SAMHD1 mutants harboring non-synonymous single nucleotide polymorphisms (SNPs) that have been identified in patients with AML, READ, STAD or COAD and performed differential analyses of ara-C sensitivity as well as restriction activity towards HIV-1 infection to unravel possible mechanistic differences in both activities. In this respect neutralization of ara-C induced cytotoxicity was found to be a very good surrogate for the enzymatic dNTPase activity of SAMHD1, and using naturally occurring SAMHD1 variants ensured that these proteins were not artificially defective, hence for the first time a direct comparison of enzymatic activity and anti-HIV-1 activity could be investigated in the same cells. We are currently investigating the effects of these SAMHD1 SNPs on oligomerization and sensitivity for degradation by Vpx. The identification of SAMHD1 SNPs altering the sensitivity to certain anti-cancer chemotherapies could also be a key for future personalized treatment strategies. Furthermore, the ability of our assays to uncouple SAMHD1 enzymatic activity from virus restriction could help to understand the contribution of SAMHD1’s dNTPase activity towards HIV-1 restriction and since the Vpx-induced rescue of HIV-1 infection from the type I IFN induced block was shown to depend on SAMHD1, may help to unravel the IFN-induced early blocks against HIV-1. In Summary, SAMHD1 plays a bigger role in the type I IFN-induced block than currently is appreciated and further investigation of its cellular function may provide insights into the underlying mechanisms and contributing additional factors.