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Nucleos(t)ide analogues are commonly used in the treatment of infectious disease and cancer. SAMHD1 is a deoxyribonucleotide (dNTP) triphosphohydrolase which is involved in the regulation of the intracellular dNTP pool, whose function has been linked to viral restriction, cancer development, and autoimmune disorders. Here, we evaluate SAMHD1 function on the antiviral and antiproliferative efficacy of a wide range of nucleos(t)ide analogues which are currently used to treat infections and cancer. The anti-HIV-1 and cytotoxic activity of compounds was assessed in primary and established cell lines in the presence or absence of SAMHD1. SAMHD1 effectively modified the anti-HIV-1 activity of all the nucleos(t)ide analogues tested, whereas sensitivity to a non-nucleoside inhibitor (nevirapine) or nucleoside phosphonates (cidofovir and tenofovir) was not affected. Interestingly, SAMHD1 could either enhance (gemcitabine, capecitabine, fluorouracil, and floxuridine) or inhibit (Ara-C, fludarabine, cladribine, clofarabine, and nelarabine) the antiviral potency of anticancer analogues, an effect that was not dependent on the specific nucleotide targeted. When cytotoxicity was evaluated, SAMHD1-dependent changes were less evident and were restricted to the increased efficacy of fluorouracil and floxuridine and reduced efficacy of nelarabine and ara-C in the presence of SAMHD1. In summary, our results demonstrate that SAMHD1 modifies the efficacy of a wide variety of nucleoside analogues which are used to treat infections, cancer, and other diseases. In addition, the anti-HIV activity of nucleos(t)ide analogues may represent a more sensitive measure of SAMHD1’s impact on drug efficacy. Thus, modulation of SAMHD1’s function may constitute a promising target for the improvement of multiple therapies.

Viral infection induces innate intracellular antiviral defenses, aimed at restricting virus replication and spread. Therefore, understanding the role and function of innate immune modulators can help to establish novel strategies for viral control. Here, we explore the role of ADAR1 as a regulator of the HIV, HCV, and HPV infections, both in vitro and in vivo, in a genetic association study. Depletion of ADAR1 induced innate immune activation, observed by a significant increase in IFNB1 mRNA and CXCL10 expression. Further characterization of ADAR1 knockdown also showed upregulation of the RNA sensors MDA5 and RIG-I, increased IRF7 expression, and phosphorylation of STAT1. ADAR1 deficiency had differential effects depending on the virus tested: siADAR1 cells showed a significant reduction in HIV-1 infection, whereas ADAR1 knockdown suggested a proviral role in HCV and HPV infections. In addition, genetic association studies were performed in a cohort of 155 HCV/HIV individuals with chronic coinfection, and a cohort of 173 HPV/HIV-infected individuals was followed for a median of six years (range 0.1–24). Polymorphisms within the ADAR1 gene were found to be significantly associated with poor clinical outcome of HCV therapy and advanced liver fibrosis in a cohort of HCV/HIV-1-coinfected patients. Moreover, we identified the low-frequency haplotype AACCAT to be significantly associated with recurrent HPV dysplasia, suggesting a role for ADAR1 in the outcome of HPV infection in HIV+ individuals. In conclusion, we show that ADAR1 regulates innate immune activation and plays a key role in susceptibility to viral infections by either limiting or enhancing viral replication. Overall, ADAR1 could be a potential target for designing immune-modulating therapeutic strategies.

Infection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1–24). We identified the low frequency haplotype AACCAT significantly associated with recurrent HPV dysplasia, suggesting a role of ADAR1 in the outcome of HPV infection in HIV+ individuals. In summary, our results suggest that ADAR1-mediated innate immune activation may influence HPV disease outcome, therefore indicating that modification of innate immune effectors regulated by ADAR1 could be a therapeutic strategy against HPV infection.

Midostaurin is a multi-kinase inhibitor with antineoplastic activity. We assessed the capacity of midostaurin to affect early and late steps of HIV-1 infection and to reactivate HIV-1 latently infected cells, alone or in combination with histone deacetylase inhibitors (HDACi) known to act as latency-reversing agents (LRA). Acute HIV-1 infection was assessed by flow cytometry in three cell types treated with midostaurin in the presence or absence of SAMHD1. Non-infected cells were treated with midostaurin and harvested for Western blot analysis. Macrophage infections were also measured by quantitative RT-PCR. HIV-1 latency reactivation was assessed in several latency models. Midostaurin induced G2/M arrest and inhibited CDK2, preventing the phosphorylation of SAMHD1 associated to inhibition of its dNTPase activity. In the presence of SAMHD1, midostaurin blocked HIV-1 DNA formation and viral replication. However, following Vpx-mediated SAMHD1 degradation, midostaurin increased viral transcripts and virus replication. In three out of four HIV-1 latency models, including primary CD4(+) T cells, midostaurin effectively reversed HIV-1 latency and was synergistic in combination with LRA vorinostat and panobinostat. Our study describes a dual effect for midostaurin in HIV-1 infection, antiviral or proviral depending on SAMHD1 activation, and highlights a role for active SAMHD1 in regulating the activity of potential HIV-1 latency reversal agents.

OBJECTIVES Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) has been shown to restrict retroviruses and DNA viruses by decreasing the pool of intracellular deoxynucleotides. In turn, SAMHD1 is controlled by cyclin-dependent kinases (CDK) that regulate the cell cycle and cell proliferation. Here, we explore the effect of CDK6 inhibitors on the replication of herpes simplex virus type 1 (HSV-1) in primary monocyte-derived macrophages (MDM). METHODS MDM were treated with palbociclib, a selective CDK4/6 inhibitor, and then infected with a GFP-expressing HSV-1. Intracellular deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. RESULTS CDK6 inhibitor palbociclib blocked SAMHD1 phosphorylation, intracellular dNTP levels and HSV-1 replication in MDM at subtoxic concentrations. Treatment of MDM with palbociclib reduced CDK2 activation, measured as the phosphorylation of the T-loop at Thr160. The antiviral activity of palbociclib was lost when SAMHD1 was degraded by viral protein X. Similarly, palbociclib did not block HSV-1 replication in SAMHD1-negative Vero cells at subtoxic concentrations, providing further evidence for a role of SAMHD1 in mediating the antiviral effect. CONCLUSIONS SAMHD1-mediated HSV-1 restriction is controlled by CDK and points to a preferential role for CDK6 and CDK2 as mediators of SAMHD1 activation. Similarly, the restricting activity of SAMHD1 against DNA viruses suggests that control of dNTP availability is the major determinant of its antiviral activity. This is the first study describing the anti-HSV-1 activity of palbociclib.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase involved in the regulation of the intracellular dNTP pool, linked to viral restriction, cancer development and autoimmune disorders. SAMHD1 function is regulated by phosphorylation through a mechanism controlled by cyclin-dependent kinases and tightly linked to cell cycle progression. Recently, SAMHD1 has been shown to decrease the efficacy of nucleotide analogs used as chemotherapeutic drugs. Here, we demonstrate that SAMHD1 can enhance or decrease the efficacy of various classes of anticancer drug, including nucleotide analogues, but also anti-folate drugs and CDK inhibitors. Importantly, we show that selective CDK4/6 inhibitors are pharmacological activators of SAMHD1 that act by inhibiting its inactivation by phosphorylation. Combinations of a CDK4/6 inhibitor with nucleoside or folate antimetabolites potently enhanced drug efficacy, resulting in highly synergic drug combinations (CI0.04). Mechanistic analyses reveal that cell cycle-controlled modulation of SAMHD1 function is the central process explaining changes in anticancer drug efficacy, therefore providing functional proof of the potential of CDK4/6 inhibitors as a new class of adjuvants to boost chemotherapeutic regimens. The evaluation of SAMHD1 expression in cancer tissues allowed for the identification of cancer types that would benefit from the pharmacological modulation of SAMHD1 function. In conclusion, these results indicate that the modulation of SAMHD1 function may represent a promising strategy for the improvement of current antimetabolite-based treatments.

Dysfunction of mitochondrial gene expression, caused by mutations in either the mitochondrial or nuclear genomes, is associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism. For instance, pathogenic mutations have been identified in the genes encoding enzymes involved in the precursor transcript processing, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, which is responsible for endonucleolytic cleavage of the 3’ ends of mitochondrial pre-tRNAs. Here, we report the identification of sixteen novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy and lactic acidosis. We provided further evidence for the pathogenicity of the three previously reported variants by studying the RNase Z activity in an in vitro system and applied this recombinant system to investigate all novel missense variants, confirming the pathogenic role of these new ELAC2 mutations. We also modelled the residues affected by missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the individuals with novel ELAC2 variants have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of hypertrophic cardiomyopathy and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, possibly indicating a functional link between tumorigenesis and mitochondrial RNA metabolism.

CD32 has been shown to be preferentially expressed in latently HIV-1-infected cells in an in vitro model of quiescent CD4 T cells. Here we show that stimulation of CD4+ T cells with IL-2, IL-7, PHA, and anti-CD3/CD28 antibodies induces T-cell proliferation, co-expression of CD32 and the activation of the markers HLA-DR and CD69. HIV-1 infection increases CD32 expression. 79.2% of the CD32+/CD4+ T cells from HIV+ individuals under antiretroviral treatment were HLA-DR+. Resting CD4+ T cells infected in vitro generally results in higher integration of provirus. We observe no difference in provirus integration or replication-competent inducible latent HIV-1 in CD32+ or CD32− CD4+ T cells from HIV+ individuals. Our results demonstrate that CD32 expression is a marker of CD4+ T cell activation in HIV+ individuals and raises questions regarding the immune resting status of CD32+ cells harboring HIV-1 proviruses., CD32 has been previously shown to be expressed preferentially by CD4 T cells latently harbouring HIV-1. Here the authors show that CD32 expression in CD4 T cells is associated with T cell activation, is up-regulated by HIV-1 infection and importantly does not appear to represent an enriched cellular niche for latent HIV-1.

The hepatitis C virus (HCV) is a globally prevalent infectious pathogen. As many as 80% of people infected with HCV do not control the virus and develop a chronic infection. Response to interferon (IFN) therapy is widely variable in chronic HCV infected patients, suggesting that HCV has evolved mechanisms to suppress and evade innate immunity responsible for its control and elimination. Adenosine deaminase acting on RNA 1 (ADAR1) is a relevant factor in the regulation of the innate immune response. The loss of ADAR1 RNA-editing activity and the resulting loss of inosine bases in RNA are critical in producing aberrant RLR-mediated innate immune response, mediated by RNA sensors MDA5 and RIG-I. Here, we describe ADAR1 role as a regulator of innate and antiviral immune function in HCV infection, both in vitro and in patients. Polymorphisms within ADAR1 gene were found significantly associated to poor clinical outcome to HCV therapy and advanced liver fibrosis in a cohort of HCV and HIV-1 coinfected patients. Moreover, ADAR1 knockdown in primary macrophages and Huh7 hepatoma cells enhanced IFN and IFN stimulated gene expression and increased HCV replication in vitro. Overall, our results demonstrate that ADAR1 regulates innate immune signaling and is an important contributor to the outcome of the HCV virus-host interaction. ADAR1 is a potential target to boost antiviral immune response in HCV infection.

The enzyme tRNase Z, a member of the metallo-beta-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase Z(S), functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase Z(L), related to tRNase Z(S) through tandem duplication and found only in eukaryotes, possesses similar to 2,000-fold greater catalytic efficiency than tRNase Z(S). tRNase Z(L) consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and T-flex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F-329 and L-332) at the carboxy end of N(dom)T(pro)x show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the N-dom-linker boundary. The Ala substitution for N-378 in the T-flex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R-382 reduces catalytic efficiency similar to 50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.

ADAR1-dependent A-to-I editing has recently been recognized as a key process for marking dsRNA as self, therefore, preventing innate immune activation and affecting the development and resolution of immune-mediated diseases and infections. Here, we have determined the role of ADAR1 as a regulator of innate immune activation and modifier of viral susceptibility in primary myeloid and lymphoid cells. We show that ADAR1 knockdown significantly enhanced interferon, cytokine and chemokine production in primary macrophages that function as antiviral paracrine factors, rendering them resistant to HIV-1 infection. ADAR1 knockdown induced deregulation of the RLRs-MAVS signaling pathway, by increasing MDA5, RIG-I, IRF7 and phospho-STAT1 expression, an effect that was partially rescued by pharmacological blockade of the pathway. In summary, our results demonstrate a role of ADAR1 in regulating innate immune function in primary macrophages, suggesting that macrophages may play an essential role in disease associated to ADAR1 dysfunction. We also show that viral inhibition is exclusively dependent on innate immune activation consequence of ADAR1 knockdown, pointing towards ADAR1 as a potential target to boost antiviral immune response.

The persistence of HIV despite suppressive antiretroviral therapy is a major roadblock to HIV eradication. Current strategies focused on inducing the expression of latent HIV fail to clear the persistent reservoir, prompting the development of new approaches for killing HIV-positive cells. Recently, acitretin was proposed as a pharmacological enhancer of the innate cellular defense network that led to virus reactivation and preferential death of infected cells. We evaluated the capacity of acitretin to reactivate and/or to facilitate immune-mediated clearance of HIV-positive cells. Acitretin did not induce HIV reactivation in latently infected cell lines (J-Lat and ACH-2). We could observe only modest induction of HIV reactivation by acitretin in latently green fluorescent protein-HIV-infected Jurkat cells, comparable to suboptimal concentrations of vorinostat, a known latency-reversing agent (LRA). Acitretin induction was insignificant, however, compared to optimal concentrations of LRAs. Acitretin failed to reactivate HIV in a model of latently infected primary CD4 + T cells but induced retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) expression in infected and uninfected cells, confirming the role of acitretin as an innate immune modulator. However, this effect was not associated with selective killing of HIV-positive cells. In conclusion, acitretin-mediated stimulation of the RIG-I pathway for HIV reactivation is modest and thus may not meaningfully affect the HIV reservoir. Stimulation of the RIG-I-dependent interferon (IFN) cascade by acitretin may not significantly affect the selective destruction of latently infected HIV-positive cells.

SAMHD1 is a triphosphohydrolase that restricts HIV-1 by limiting the intracellular dNTP pool required for reverse transcription. Although SAMHD1 is expressed and active/unphosphorylated in most cell lines, its restriction activity is thought to be relevant only in non-cycling cells. However, an in depth evaluation of SAMHD1 function and relevance in cycling cells is required. Here, we show that SAMHD1-induced degradation by HIV-2 Vpx affects the dNTP pool and HIV-1 replication capacity in the presence of the 3′-azido-3′-deoxythymidine (AZT) in cycling cells. Similarly, in SAMHD1 knockout cells, HIV-1 showed increased replicative capacity in the presence of nucleoside inhibitors, especially AZT, that was reverted by re-expression of wild type SAMHD1. Sensitivity to non-nucleoside inhibitors (nevirapine and efavirenz) or the integrase inhibitor raltegravir was not affected by SAMHD1. Combination of three mutations (S18A, T21A, T25A) significantly prevented SAMHD1 phosphorylation but did not significantly affect HIV-1 replication in the presence of AZT. Our results demonstrate that SAMHD1 is active in HIV-1 permissive cells, does not modify susceptibility to HIV-1 infection but strongly affects sensitivity to nucleoside inhibitors., Spanish Ministerio de Economía y Competitividad (MINECO) project BFU2015-63800-R and BIO2013-48788-C2-1-R and Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (FIS) PI13/01083, PI15/00492 and PI16/00103 cofinanced by FEDER

HIV-1 infection is thought to impair type I interferon (IFN-I) production in macrophages, a cell type that is also relatively resistant to HIV-1 cytotoxic effects. Here, we show that monocyte differentiation into macrophages by M-CSF led to cell proliferation and susceptibility to HIV-1 infection that induced cell cycle arrest and increased cell death. Established HIV-1 infection of monocyte-derived macrophages induced the upregulation of the pattern recognition receptors MDA5 and Rig-I that serve as virus sensors; production of interferon-β, and transcription of interferon-stimulated genes including CXCL10. Infected macrophages showed increased expression of p21 and subsequent inactivation of cyclin-CDK2 activity leading to a hypo-phosphorylated active retinoblastoma protein (pRb) and deactivation of E2F1-dependent transcription and CDK1 downregulation. Additionally, HIV-1 infection limited deoxynucleotide pool by downregulation of the ribonucleotide reductase subunit R2 (RNR2) and reactivation of the HIV-1 restriction factor SAMHD1 together with increased cell death. In conclusion, HIV-1 induced an innate antiviral mechanism associated to IFN-I production, interferon stimulated gene activation, and p21-mediated G2/M arrest leading to elevated levels of cell death in monocyte derived macrophages. Upregulation of MDA5 and Rig-I may serve as targets for the development of antiviral strategies leading to the elimination of HIV-1 infected cells.

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages., Author Summary Macrophages are a heterogeneous population of immune cells that provide crucial innate immune defense against pathogens, including HIV-1. The molecular biology of HIV-1 infection in macrophages is influenced by the presence of the host cell restriction factor SAMHD1, which is regulated by phosphorylation by cyclin dependent kinases, the catalytic proteins responsible for cell cycle progression. This study shows that differentiation stimuli strongly influence macrophage cell cycle and proliferation characteristics as well as susceptibility to HIV-1 infection through modulation of SAMHD1 activation. We have identified cyclin D2 as the key step controlling susceptibility to HIV-1 infection by modulation of the signaling pathway leading to SAMHD1 phosphorylation. We show that a complex formed by cyclin D2-CDK4-p21 in GM-CSF macrophages is responsible for the lack of the active CDK, which phosphorylates SAMHD1. This situation is reversed in the absence of cyclin D2, leading to the activation of CDKs and subsequent phosphorylation of its substrates, including SAMHD1. Thus, we propose that the differential expression of the G1/S-specific cyclin D2 controls the HIV-1 restriction pathway in primary macrophages.