Targeting small GTPases and their downstream pathways with intracellular macromolecule binders to define alternative therapeutic strategies in cancer.

The RAS superfamily of small GTPases regulates major physiological cellular processes. Mutation or deregulation of these small GTPases, their regulators and/or their effectors are associated with many diseases including cancer. Hence, targeting these classes of proteins is an important therapeutic strategy in cancer. This has been recently achieved with the approval of the first KRASG12C covalent inhibitors for the clinic. However, many other mutants and small GTPases are still considered as 'undruggable' with small molecule inhibitors because of a lack of well-defined pocket(s) at their surface. Therefore, alternative therapeutic strategies have been developed to target these proteins. In this review, we discuss the use of intracellular antibodies and derivatives - reagents that bind their antigen inside the cells - for the discovery of novel inhibitory mechanisms, targetable features and therapeutic strategies to inhibit small GTPases and their downstream pathways. These reagents are also versatile tools used to better understand the biological mechanisms regulated by small GTPases and to accelerate the drug discovery process.

Mediation of Interleukin-23 and Tumor Necrosis Factor-Driven Reactive Arthritis by Chlamydia-Infected Macrophages in SKG Mice.

OBJECTIVE: ZAP-70W163C BALB/c (SKG) mice develop reactive arthritis (ReA) following infection with Chlamydia muridarum. Since intracellular pathogens enhance their replicative fitness in stressed host cells, we examined how myeloid cells infected with C muridarum drive arthritis. METHODS: SKG, Il17a-deficient SKG, and BALB/c female mice were infected with C muridarum or C muridarum luciferase in the genitals. C muridarum dissemination was assessed by in vivo imaging or genomic DNA amplification. Macrophages were depleted using clodronate liposomes. Anti-tumor necrosis factor (anti-TNF) and anti-interleukin-23p19 (anti-IL-23p19) were administered after infection or arthritis onset. Gene expression of Hspa5, Tgtp1, Il23a, Il17a, Il12b, and Tnf was compared in SKG mice and BALB/c mice. RESULTS: One week following infection with C muridarum, macrophages and neutrophils were observed to have infiltrated the uteri of mice and were also shown to have carried C muridarum DNA to the spleen. C muridarum load was higher in SKG mice than in BALB/c mice. Macrophage depletion was shown to reduce C muridarum load and prevent development of arthritis. Compared with BALB/c mice, expression of Il23a and Il17a was increased in the uterine and splenic neutrophils of SKG mice. The presence of anti-IL-23p19 during infection or Il17a deficiency suppressed arthritis. Tnf was overexpressed in the joints of SKG mice within 1 week postinfection, and persisted beyond the first week. TNF inhibition during infection or at arthritis onset suppressed the development of arthritis. Levels of endoplasmic reticulum stress were constitutively increased in the joints of SKG mice but were induced, in conjunction with immunity-related GTPase, by C muridarum infection in the uterus. CONCLUSION: C muridarum load is higher in SKG mice than in BALB/c mice. Whereas proinflammatory IL-23 produced by neutrophils contributes to the initiation of C muridarum-mediated ReA, macrophage depletion reduces C muridarum dissemination to other tissues, tissue burden, and the development of arthritis. TNF inhibition was also shown to suppress arthritis development. Our data suggest that enhanced bacterial dissemination in macrophages of SKG mice drives the TNF production needed for persistent arthritis.

Nutrient mTORC1 signaling underpins regulatory T cell control of immune tolerance.

Foxp3+ regulatory T (T reg) cells are pivotal regulators of immune tolerance, with T cell receptor (TCR)-driven activated T reg (aT reg) cells playing a central role; yet how TCR signaling propagates to control aT reg cell responses remains poorly understood. Here we show that TCR signaling induces expression of amino acid transporters, and renders amino acid-induced activation of mTORC1 in aT reg cells. T reg cell-specific ablation of the Rag family small GTPases RagA and RagB impairs amino acid-induced mTORC1 signaling, causing defective amino acid anabolism, reduced T reg cell proliferation, and a rampant autoimmune disorder similar in severity to that triggered by T reg cell-specific TCR deficiency. Notably, T reg cells in peripheral tissues, including tumors, are more sensitive to Rag GTPase-dependent nutrient sensing. Ablation of RagA alone impairs T reg cell accumulation in the tumor, resulting in enhanced antitumor immunity. Thus, nutrient mTORC1 signaling is an essential component of TCR-initiated T reg cell reprogramming, and Rag GTPase activities may be titrated to break tumor immune tolerance.

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating 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.

SAMHD1 transcript upregulation during SIV infection of the central nervous system does not associate with reduced viral load.

Restriction of HIV-1 in myeloid-lineage cells is attributed in part to the nucleotidase activity of the SAM-domain and HD-domain containing protein (SAMHD1), which depletes free nucleotides, blocking reverse transcription. In the same cells, the Vpx protein of HIV-2 and most SIVs counteracts SAMHD1. Both Type I and II interferons may stimulate SAMHD1 transcription. The contributions of SAMHD1 to retroviral restriction in the central nervous system (CNS) have been the subject of limited study. We hypothesized that SAMHD1 would respond to interferon in the SIV-infected CNS but would not control virus due to SIV Vpx. Accordingly, we investigated SAMHD1 transcript abundance and association with the Type I interferon response in an SIV model. SAMHD1 transcript levels were IFN responsive, increasing during acute phase infection and decreasing during a more quiescent phase, but generally remaining elevated at all post-infection time points. In vitro, SAMHD1 transcript was abundant in macaque astrocytes and further induced by Type I interferon, while IFN produced a weaker response in the more permissive environment of the macrophage. We cannot rule out a contribution of SAMHD1 to retroviral restriction in relatively non-permissive CNS cell types. We encourage additional research in this area, particularly in the context of HIV-1 infection.

Repression of Mammalian Target of Rapamycin Complex 1 Inhibits Intestinal Regeneration in Acute Inflammatory Bowel Disease Models.

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues to regulate cell growth and survival through various mechanisms. However, how mTORC1 responds to acute inflammatory signals to regulate bowel regeneration is still obscure. In this study, we investigated the role of mTORC1 in acute inflammatory bowel disease. Inhibition of mTORC1 activity by rapamycin treatment or haploinsufficiency of Rheb through genetic modification in mice impaired intestinal cell proliferation and induced cell apoptosis, leading to high mortality in dextran sodium sulfate- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis models. Through bone marrow transplantation, we found that mTORC1 in nonhematopoietic cells played a major role in protecting mice from colitis. Reactivation of mTORC1 activity by amino acids had a positive therapeutic effect in mTORC1-deficient Rheb(+/-) mice. Mechanistically, mTORC1 mediated IL-6-induced Stat3 activation in intestinal epithelial cells to stimulate the expression of downstream targets essential for cell proliferation and tissue regeneration. Therefore, mTORC1 signaling critically protects against inflammatory bowel disease through modulation of inflammation-induced Stat3 activity. As mTORC1 is an important therapeutic target for multiple diseases, our findings will have important implications for the clinical usage of mTORC1 inhibitors in patients with acute inflammatory bowel disease.

Inhibition of CD40-induced N-Ras activation reduces leishmania major infection.

Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major-altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major-expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras-Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major-enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform-targeted antileishmanial immunotherapy and immunoprophylaxis.

Phenotypic variation in Aicardi-Goutières syndrome explained by cell-specific IFN-stimulated gene response and cytokine release.

Aicardi-Goutières syndrome (AGS) is a monogenic inflammatory encephalopathy caused by mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, or MDA5. Mutations in those genes affect normal RNA/DNA intracellular metabolism and detection, triggering an autoimmune response with an increase in cerebral IFN-α production by astrocytes. Microangiopathy and vascular disease also contribute to the neuropathology in AGS. In this study, we report that AGS gene silencing of TREX1, SAMHD1, RNASEH2A, and ADAR1 by short hairpin RNAs in human neural stem cell-derived astrocytes, human primary astrocytes, and brain-derived endothelial cells leads to an antiviral status of these cells compared with nontarget short hairpin RNA-treated cells. We observed a distinct activation of the IFN-stimulated gene signature with a substantial increase in the release of proinflammatory cytokines (IL-6) and chemokines (CXCL10 and CCL5). A differential impact of AGS gene silencing was noted; silencing TREX1 gave rise to the most dramatic in both cell types. Our findings fit well with the observation that patients carrying mutations in TREX1 experience an earlier onset and fatal outcome. We provide in the present study, to our knowledge for the first time, insight into how astrocytic and endothelial activation of antiviral status may differentially lead to cerebral pathology, suggesting a rational link between proinflammatory mediators and disease severity in AGS.

Distinct characteristics of endometrial and decidual macrophages and regulation of their permissivity to HIV-1 infection by SAMHD1.

UNLABELLED: In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages are the main HIV-1 target cells in the decidua. Here we report that in vitro, macrophages and T cells are the main HIV-1 targets in the endometrium in nonpregnant women. As reported for decidual macrophages (dM), endometrial macrophages (eM) were found to have an M2-like phenotype (CD68+ CD163+ CD206+ IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of proinflammatory and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role for SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with FRT mucosae in nonpregnant women should help to identify mechanisms of mucosal protection against HIV-1 infection. IMPORTANCE: The female reproductive tract mucosae are major portals of HIV-1 entry into the body. The decidua (uterine mucosa during pregnancy) can serve as a model for studying natural mucosal protection against HIV-1 transmission. A comparison of target cells and innate responses in the decidua versus the endometrium in nonpregnant women could help to identify protective mechanisms. Here, we report for the first time that macrophages are one of the main HIV-1 target cells in the endometrium and that infection of macrophages from both the endometrium and the decidua is restricted by SAMHD1. These findings might have implications for the development of vaccines to prevent HIV-1 mucosal transmission.

Development of monoclonal antibodies specifically recognizing the endogenous sterile alpha motif and HD domain 1 protein in porcine cell lines.

The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor that participates in processes crucial to the viral life cycle. In the present study, we describe a procedure to generate monoclonal antibodies (MAbs) against porcine SAMHD1 and investigate its characteristics to analyze the expression of endogenous SAMHD1. The open reading frame of porcine SAMHD1 was cloned into the prokaryotic expression vector pCold-TF DNA to construct a recombinant plasmid pcold-pSAMHD1 and induce expression of recombinant porcine SAMHD1 protein by IPTG in Escherichia coli Rosetta. The purified recombinant porcine SAMHD1 protein was used to prepare MAbs of SAMHD1. After subcloning five times hybridoma cell clones expressing SAMHD1, MAbs were generated. Western blot analysis and indirect immunofluorescence assay showed that the overexpressed porcine SAMHD1 in 293T cells and endogenous SAMHD1 protein in porcine cell lines could be specifically recognized by the MAbs produced in this study. In conclusion, specific MAbs of porcine SAMHD1 are reported, and these MAbs provide a valuable tool for further studies of SAMHD1-mediated signaling in virus-infected cells to elucidate the underlying antiviral mechanism.

Unmasking immune sensing of retroviruses: interplay between innate sensors and host effectors.

Retroviruses can selectively trigger an array of innate immune responses through various PRR. The identification and the characterization of the molecular basis of retroviral DNA sensing by the DNA sensors IFI16 and cGAS has been one of the most exciting developments in viral immunology in recent years. DNA sensing by these cytosolic sensors not only leads to the initiation of the type I interferon (IFN) antiviral response and the induction of the inflammatory response, but also triggers cell death mechanisms including pyroptosis and apoptosis in retrovirus-infected cells, thereby providing important insights into the pathophysiology of chronic retroviral infection. Host restriction factors such as SAMHD1 and Trex1 play important roles in regulating innate immune sensing, and have led to the idea that innate immune defense and host restriction actually converge at different levels to determine the outcome of retroviral infection. In this review, we discuss the sensing of retroviruses by cytosolic DNA sensors, the relevance of host factors during retroviral infection, and the interplay between host factors and the innate antiviral response in different cell types, within the context of two human pathogenic retroviruses - human immunodeficiency virus (HIV-1) and human T cell-leukemia virus type I (HTLV-1).

Molecular cloning and characterizations of porcine SAMHD1 and its roles in replication of highly pathogenic porcine reproductive and respiratory syndrome virus.

The sterile alpha motif and HD domain 1 (SAMHD1) protein is a novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity.

CD4+ memory stem cells are infected by HIV-1 in a manner regulated in part by SAMHD1 expression.

UNLABELLED: CD4(+) and CD8(+) memory T cells with stem cell-like properties (T(SCM) cells) have been identified in mice, humans, and nonhuman primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4(+) T(SCM) cells are infected by human immunodeficiency virus (HIV) was investigated by using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of T(SCM) cells were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that T(SCM) cells can become productively or latently infected, although the vast majority of T(SCM) cells are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing simian immunodeficiency virus (SIV) virion-like particles enhanced the productive infection of T(SCM) cells, indicating that SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4(+) T(SCM) cells are targets for HIV infection, that they become productively or latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory cell subset. IMPORTANCE: Here we demonstrate the susceptibility of CD4(+) memory stem cells (T(SCM) cells) to infection by HIV in vitro and in vivo, provide an in-depth analysis of coreceptor expression, demonstrate the infection of naïve and memory CD4(+) T cell subsets with both CCR5- and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, or abortively infected. Through these outcome studies, we determined that the vast majority of T(SCM) cells are abortively infected by HIV, and we demonstrate that knockdown of SAMHD1 significantly increases the frequency of infection of this CD4(+) T cell subset, indicating that SAMHD1 is an active restriction factor in T(SCM) cells.

miR-155 suppresses bacterial clearance in Pseudomonas aeruginosa-induced keratitis by targeting Rheb.

miR-155 (microRNA-155) is an important noncoding RNA in regulating host inflammatory responses. However, its regulatory role in ocular infection remains unclear. Our study first explored the function of miR-155 in Pseudomonas aeruginosa-induced keratitis, one of the most common sight-threatening ocular diseases. We found that miR-155 expression was enhanced in human and mouse corneas after P. aeruginosa infection and was mainly expressed in macrophages but not neutrophils. In vivo studies demonstrated that miR-155 knockout mice displayed more resistance to P. aeruginosa keratitis, with a higher inducible nitric oxide synthase level and a lower bacterial burden. More importantly, in vitro data indicated that miR-155 suppressed the macrophage-mediated bacterial phagocytosis and intracellular killing of P. aeruginosa by targeting Rheb (Ras homolog enriched in brain). To the best of our knowledge, this is the first study to explore the role of miR-155 in bacterial keratitis, which may provide a promising target for clinical treatment of P. aeruginosa keratitis and other infectious diseases.

Host SAMHD1 protein promotes HIV-1 recombination in macrophages.

Template switching can occur during the reverse transcription of HIV-1. Deoxynucleotide triphosphate (dNTP) concentrations have been biochemically shown to impact HIV-1 reverse transcriptase (RT)-mediated strand transfer. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. Terminally differentiated/nondividing macrophages, which serve as a key HIV-1 reservoir, contain extremely low dNTP concentrations (20-50 nm), which results from the cellular dNTP hydrolyzing sterile α motif and histidine aspartic domain containing protein 1 (SAMHD1) protein, when compared with activated CD4(+) T cells (2-5 µm). In this study, we first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4(+) T cells. Second, SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4(+) T cells. Third, differentiated SAMHD1 shRNA THP-1 cells have a 2-fold increase in HIV-1 template switching efficiency. Fourth, SAMHD1 degradation by Vpx did not alter HIV-1 template switching efficiency in activated CD4(+) T cells. Finally, the HIV-1 V148I RT mutant that is defective in dNTP binding and has DNA synthesis delay promoted RT stand transfer when compared with wild type RT, particularly at low dNTP concentrations. Here, we report that SAMHD1 regulation of the dNTP concentrations influences HIV-1 template switching efficiency, particularly in macrophages.

Restriction of retroviral infection of macrophages.

Primate immunodeficiency viruses are highly specialized lentiviruses that have evolved to successfully infect and persist for the lifetime of the host. Despite encountering numerous potent antiviral factors, HIVs and SIVs are successful pathogens due to the acquisition of equally potent countermeasures in the form of accessory genes. The accessory gene Vpx encoded by HIV-2 and a subset of SIVs have a profound effect on the ability of lentiviruses to infect non-dividing cells, such as macrophages. Although most virus replication occurs in activated CD4(+) T cells, myeloid lineage cells are natural targets of infection and play a central role in virus transmission, dissemination, and persistence. However, myeloid lineage cells are poorly sensitive to lentiviral infection due partly to the high-level expression of a host protein that regulates nucleic acid metabolism named SAMHD1. Degradation of SAMHD1 is induced by Vpx to eliminate this intrinsic antiviral factor. Importantly, SAMHD1 has also been implicated as a negative regulator of the innate immune response, so the interplay between SAMHD1 and Vpx is likely to have significant consequences for virus replication, persistence, and immune control.

Restriction of HIV-1 replication in primary macrophages by IL-12 and IL-18 through the upregulation of SAMHD1.

Monocyte-derived macrophages (MDM) can polarize into different subsets depending on the environment and the activation signal to which they are submitted. Differentiation into macrophages allows HIV-1 strains to infect cells of the monocytic lineage. In this study, we show that culture of monocytes with a combination of IL-12 and IL-18 led to macrophage differentiation that was resistant to HIV-1 infection. In contrast, M-CSF-derived MDM were readily infected by HIV-1. When monocytes were differentiated in the presence of M-CSF and then further treated with IL-12/IL-18, cells became resistant to infection. The restriction on HIV-1 replication was not dependent on virus entry or coreceptor expression, as vesicular stomatitis virus-pseudotyped HIV-1 replication was also blocked by IL-12/IL-18. The HIV-1 restriction factor sterile α motif and HD domain-containing protein-1 (SAMHD1) was significantly overexpressed in IL-12/IL-18 MDM compared with M-CSF MDM, and degradation of SAMHD1 by RNA interference or viral-like particles carrying the lentiviral protein Vpx restored HIV-1 infectivity of IL-12/IL-18 MDM. SAMHD1 overexpression induced by IL-12/IL-18 was not dependent on IFN-γ. Thus, we conclude that IL-12 and IL-18 may contribute to the response against HIV-1 infection through the induction of restriction factors such as SAMHD1.

Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply.

BACKGROUND: Interferon-α (IFN-α) is an essential mediator of the antiviral response, which potently inhibits both early and late phases of HIV replication. The SAMHD1 deoxynucleoside triphosphate (dNTP) hydrolase represents the prototype of a new antiviral strategy we referred to as "nucleotide depletion". SAMHD1 depletes dNTP levels in myeloid cells below those required for optimal synthesis of HIV viral DNA. HIV-2 and its SIVsm and SIVmac close relatives encode a protein termed Vpx, which counteracts SAMHD1. The potentiality of IFN-α to cooperate with nucleotide depletion has been poorly investigated so far. Here we wondered whether IFN-α affects SAMHD1 expression, Vpx-induced SAMHD1 degradation, Vpx-mediated rescue of HIV-1 transduction and the dNTP supply in monocyte-derived macrophages (MDMs). RESULTS: IFN-α inhibited HIV-1 transduction in monocytes and in MDMs while SAMHD1 expression was not up-regulated. Vpx triggered SAMHD1 degradation in IFN-α treated cells, and weakly restored HIV-1 transduction from the IFN-α block. Vpx helper effect towards HIV-1 transduction was gradually inhibited with increasing doses of IFN-α. dNTP levels were not significantly affected in MDMs and CD4+ primary activated T lymphocytes by IFN-α and, in correlation with SAMHD1 degradation, restoration of dNTP levels by Vpx was efficient in MDMs treated with the cytokine. In contrast, IFN-α inhibited Vpx-mediated SAMHD1 degradation in THP-1 cells, where, accordingly, Vpx could not rescue HIV-1 transduction. CONCLUSION: Our results suggest that the early antiviral effect of IFN-α results from a mechanism independent of nucleotide depletion in MDMs. In addition, they indicate that the macrophage-like THP-1 cell line may provide a system to characterize an IFN-α-induced cell response that inhibits Vpx-mediated SAMHD1 degradation.

Activation of the small G-protein Rac by human rhinovirus attenuates the TLR3/IFN-α axis while promoting CCL2 release in human monocyte-lineage cells.

Although rhinoviral infections, a major cause of asthma exacerbations, occur predominantly in upper airway bronchial epithelial cells, monocytic-lineage cells are implicated in establishing the inflammatory microenvironment observed during the disease. Human rhinovirus (HRV) is unique in that nearly genetically identical viruses bind either the ICAM-1 or low-density lipoprotein receptor (LDL-R). Within minutes of binding, HRV is capable of eliciting a signaling response in both epithelial cells and monocyte-derived macrophages. It is unclear whether this signaling response is important to the subsequent release of inflammatory mediators, particularly in cells not capable of supporting viral replication. We show here that the small molecular mass G-protein Rac is activated following exposure of macrophages to HRV serotypes known to be ICAM-1- and LDL-R-tropic. We demonstrate that inhibiting Rac resulted in the upregulation of TLR3 in macrophages exposed to major- and minor-group HRV, and resulted in increased release of IFN-α. Furthermore, inhibiting Rac in HRV-exposed macrophages attenuated activation of the stress kinase p38 and release of the pro-inflammatory cytokine CCL2, but inhibiting Rac did not affect release of the pro-inflammatory cytokine CCL5. These findings suggest that Rac is an important regulator in establishing the inflammatory microenvironment that is initiated in the human airway upon exposure to rhinovirus.

Intrinsic cellular defenses against human immunodeficiency viruses.

Viral infections are often detrimental to host survival and reproduction. Consequently, hosts have evolved a variety of mechanisms to defend themselves against viruses. A component of this arsenal is a set of proteins, termed restriction factors, which exhibit direct antiviral activity. Among these are several classes of proteins (APOBEC3, TRIM5, Tetherin, and SAMHD1) that inhibit the replication of human and simian immunodeficiency viruses. Here, we outline the features, mechanisms, and evolution of these defense mechanisms. We also speculate on how restriction factors arose, how they might interact with the conventional innate and adaptive immune systems, and how an understanding of these intrinsic cellular defenses might be usefully exploited.