Dissecting SAMHD1´s role in the type I Interferon induced early block to HIV-1 infection and its connection to cancer

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

Effects of inner nuclear membrane proteins SUN1/UNC-84A and SUN2/UNC- 84B on the early steps of HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1NL4.3 and HIV-1IIIB) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro-assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1−/− and SUN2−/− cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection. IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis

The role of inner nuclear envelope associated proteins SUN1 and SUN2 in early HIV-1 infection steps

status: submitte

Complex interplay between HIV-1 capsid and MX2-independent alpha interferon-induced antiviral factors

Type I interferons (IFNs), including IFN-α, upregulate an array of IFN-stimulated genes (ISGs) and potently suppress Human immunodeficiency virus type 1 (HIV-1) infectivity in CD4(+) T cells, monocyte-derived macrophages, and dendritic cells. Recently, we and others identified ISG myxovirus resistance 2 (MX2) as an inhibitor of HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection, e.g., prior to (or during) reverse transcription, remain to be defined. We show here that the HIV-1 CA mutations N74D and A105T, both of which allow escape from inhibition by MX2 and the truncated version of cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutation P90A, all increase sensitivity to IFN-α-mediated inhibition. Using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, we demonstrate that the IFN-α hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant but modestly increased the IFN-α sensitivity of wild-type virus. Interestingly, the infectivity of wild-type or P90A virus could be rescued from the MX2-independent IFN-α-induced blocks in THP-1 cells by treatment with cyclosporine (Cs) or its nonimmunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFN-α-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shield the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection