scholarly journals p21 Restricts HIV-1 in Monocyte-Derived Dendritic Cells through the Reduction of Deoxynucleoside Triphosphate Biosynthesis and Regulation of SAMHD1 Antiviral Activity

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Jose Carlos Valle-Casuso ◽  
Awatef Allouch ◽  
Annie David ◽  
Gina M. Lenzi ◽  
Lydia Studdard ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dendritic Cells ◽  
Antiviral Activity ◽  
Immune Responses ◽  
Reverse Transcription ◽  
Kinase Inhibitor ◽  
Cell Model ◽  
Dntp Pool ◽  
Intracellular Dntp ◽  
Hiv 1

ABSTRACT HIV-1 infection of noncycling cells, such as dendritic cells (DCs), is impaired due to limited availability of deoxynucleoside triphosphates (dNTPs), which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during the cell cycle and depend on the balance between dNTP biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and noncycling cells. Here, we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated with an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replication, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1. IMPORTANCE DCs play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, potentially can contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phosphorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Thus, induction of p21 resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.

scholarly journals USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Edmund Osei Kuffour ◽  
Kerstin Schott ◽  
Ananda Ayyappan Jaguva Vasudevan ◽  
Jessica Holler ◽  
Wolfgang A. Schulz ◽  
...  
Keyword(s):  
Protein Expression ◽  
Reverse Transcription ◽  
Kinase Inhibitor ◽  
Active Form ◽  
Innate Immune ◽  
P21 Protein ◽  
Intracellular Dntp ◽  
Antiviral Function ◽  
Reverse Transcription Step ◽  
Hiv 1

ABSTRACTThe host intrinsic innate immune system drives antiviral defenses and viral restriction, which includes the production of soluble factors, such as type I and III interferon (IFN), and activation of restriction factors, including SAMHD1, a deoxynucleoside triphosphohydrolase. Interferon-stimulated gene 15 (ISG15)-specific ubiquitin-like protease 43 (USP18) abrogates IFN signaling pathways. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1), which is involved in the differentiation and maturation of monocytes, inhibits human immunodeficiency virus type 1 (HIV-1) in macrophages and dendritic cells. p21 inhibition of HIV-1 replication is thought to occur at the reverse transcription step, likely by suppressing cellular deoxynucleoside triphosphate (dNTP) biosynthesis and increasing the amount of antivirally active form of SAMHD1. SAMHD1 strongly inhibits HIV-1 replication in myeloid and resting CD4+T cells. Here, we studied how USP18 influences HIV-1 replication in human myeloid THP-1 cells. We found that USP18 has the novel ability to inhibit the antiviral function of p21 in differentiated THP-1 cells. USP18 enhanced reverse transcription of HIV-1 by downregulating p21 expression and upregulating intracellular dNTP levels. p21 downregulation by USP18 was associated with the active form of SAMHD1, phosphorylated at T592. USP18 formed a complex with the E3 ubiquitin ligase recognition factor SKP2 (S-phase kinase associated protein 2) and SAMHD1. CRISPR-Cas9 knockout of USP18 increased p21 protein expression and blocked HIV-1 replication. Overall, we propose USP18 as a regulator of p21 antiviral function in differentiated myeloid THP-1 cells.IMPORTANCEMacrophages and dendritic cells are usually the first point of contact with pathogens, including lentiviruses. Host restriction factors, including SAMHD1, mediate the innate immune response against these viruses. However, HIV-1 has evolved to circumvent the innate immune response and establishes disseminated infection. The cyclin-dependent kinase inhibitor p21, which is involved in differentiation and maturation of monocytes, blocks HIV-1 replication at the reverse transcription step. p21 is thought to suppress key enzymes involved in dNTP biosynthesis and activates SAMHD1 antiviral function. We report here that the human USP18 protein is a novel factor potentially contributing to HIV replication by blocking the antiviral function of p21 in differentiated human myeloid cells. USP18 downregulates p21 protein expression, which correlates with upregulated intracellular dNTP levels and the antiviral inactive form of SAMHD1. Depletion of USP18 stabilizes p21 protein expression, which correlates with dephosphorylated SAMHD1 and a block to HIV-1 replication.

scholarly journals SAMHD1 … and Viral Ways around It

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 395
Author(s):  
Janina Deutschmann ◽  
Thomas Gramberg
Keyword(s):  
Dna Damage ◽  
Antiviral Activity ◽  
Immune Responses ◽  
Restriction Factor ◽  
Virus Species ◽  
Cellular Functions ◽  
Dna Viruses ◽  
Damage Repair ◽  
Intracellular Dntp ◽  
Hiv 1

The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1.

scholarly journals Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells

2017 ◽  
Vol 114 (10) ◽  
pp. 2729-2734 ◽  
Author(s):  
Hanna-Mari Baldauf ◽  
Lena Stegmann ◽  
Sarah-Marie Schwarz ◽  
Ina Ambiel ◽  
Maud Trotard ◽  
...  
Keyword(s):  
T Cells ◽  
Reverse Transcription ◽  
Cd4 T Cells ◽  
Single Amino Acid ◽  
Sooty Mangabey ◽  
Dntp Pool ◽  
Sam Domain ◽  
Virion Incorporation ◽  
Intracellular Dntp ◽  
Hiv 1

Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2–like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

scholarly journals SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charlotte Martinat ◽  
Arthur Cormier ◽  
Joëlle Tobaly-Tapiero ◽  
Noé Palmic ◽  
Nicoletta Casartelli ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcription ◽  
Immune Cells ◽  
Viral Restriction ◽  
Antiviral Function ◽  
Hiv 1 ◽  
Constitutively Active

AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

scholarly journals Inhibition of lipid antigen presentation in dendritic cells by HIV-1 Vpu interference with CD1d recycling from endosomal compartments

Blood ◽  
2010 ◽  
Vol 116 (11) ◽  
pp. 1876-1884 ◽  
Author(s):  
Markus Moll ◽  
Sofia K. Andersson ◽  
Anna Smed-Sörensen ◽  
Johan K. Sandberg
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Antigen Presentation ◽  
Nkt Cells ◽  
Cellular Immune Responses ◽  
Antigen Presentation Pathway ◽  
Early Endosomes ◽  
Presentation Pathway ◽  
Cellular Immune ◽  
Hiv 1

AbstractDendritic cells (DCs) play an important role in viral infections both as initiators of immunity and as viral targets. Interaction between DCs and the innate-like CD1d-restricted natural killer T (NKT) cells results in the mutual activation of both cells and the subsequent initiation of cellular immune responses. Here, we show that HIV-1 inhibits the surface expression of CD1d in productively infected DCs and identify this as a novel activity of the HIV-1 vpu gene product. Interestingly, the viral protein U (Vpu) does not enhance constitutive CD1d endocytosis or induce rapid CD1d degradation. Instead, the Vpu protein interacts with CD1d and suppresses its recycling from endosomal compartments to the cell surface by retaining CD1d in early endosomes. This interference with the CD1d antigen presentation pathway strongly inhibits the ability of infected DCs to activate CD1d-restricted NKT cells. Given that the interaction with CD1d-expressing DCs is central to the ability of NKT cells to regulate immunity, these data suggest that interference with the CD1d antigen presentation pathway represents an HIV-1 strategy to evade innate cellular immune responses and imply a role for the innate-like CD1d-restricted NKT cells in the host defense against HIV-1.

scholarly journals Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Caitlin M. Miller ◽  
Hisashi Akiyama ◽  
Luis M. Agosto ◽  
Ann Emery ◽  
Chelsea R. Ettinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Culture ◽  
Dendritic Cells ◽  
Ubiquitin Ligase ◽  
Culture System ◽  
E3 Ubiquitin Ligase ◽  
Cell Culture System ◽  
Ubiquitin Ligase Complex ◽  
Hiv 1

ABSTRACT Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4+ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication. IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

scholarly journals Immature Dendritic Cells Selectively Replicate Macrophagetropic (M-Tropic) Human Immunodeficiency Virus Type 1, while Mature Cells Efficiently Transmit both M- and T-Tropic Virus to T Cells

1998 ◽  
Vol 72 (4) ◽  
pp. 2733-2737 ◽  
Author(s):  
Angela Granelli-Piperno ◽  
Elena Delgado ◽  
Victoria Finkel ◽  
William Paxton ◽  
Ralph M. Steinman
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Dendritic Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Dendritic cells (DCs) can develop from CD14+ peripheral blood monocytes cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). By 6 days in culture, the cells have the characteristics of immature DCs and can be further induced to mature by inflammatory stimuli or by monocyte-conditioned medium. After infection with macrophagetropic (M-tropic) human immunodeficiency virus type 1 (HIV-1), monocytes and mature DCs show a block in reverse transcription and only form early transcripts that can be amplified with primers for the R/U5 region. In contrast, immature DCs cultured for 6 or 11 days in GM-CSF and IL-4 complete reverse transcription and show a strong signal when LTR/gag primers are used. Blood monocytes and mature DCs do not replicate HIV-1, whereas immature DCs can be productively infected, but only with M-tropic HIV-1. The virus produced by immature DCs readily infects activated T cells. Although mature DCs do not produce virus, these cells transmit both M- and T-tropic virus to T cells. In the cocultures, both DCs and T cells must express functional chemokine coreceptors for viral replication to occur. Therefore, the developmental stage of DCs can influence the interaction of these cells with HIV-1 and influence the extent to which M-tropic and T-tropic virus can replicate.

scholarly journals Anti-CD40 Antibody Fused to CD40 Ligand Is a Superagonist Platform for Adjuvant Intrinsic DC-Targeting Vaccines

2022 ◽  
Vol 12 ◽  
Author(s):  
Valentina Ceglia ◽  
Sandra Zurawski ◽  
Monica Montes ◽  
Mitchell Kroll ◽  
Aurélie Bouteau ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Immune Responses ◽  
Class I ◽  
Agonist Activity ◽  
Cell Immunity ◽  
Hiv 1

CD40 is a potent activating receptor expressed on antigen-presenting cells (APCs) of the immune system. CD40 regulates many aspects of B and T cell immunity via interaction with CD40L expressed on activated T cells. Targeting antigens to CD40 via agonistic anti-CD40 antibody fusions promotes both humoral and cellular immunity, but current anti-CD40 antibody-antigen vaccine prototypes require co-adjuvant administration for significant in vivo efficacy. This may be a consequence of dulling of anti-CD40 agonist activity via antigen fusion. We previously demonstrated that direct fusion of CD40L to anti-CD40 antibodies confers superagonist properties. Here we show that anti-CD40-CD40L-antigen fusion constructs retain strong agonist activity, particularly for activation of dendritic cells (DCs). Therefore, we tested anti-CD40-CD40L antibody fused to antigens for eliciting immune responses in vitro and in vivo. In PBMC cultures from HIV-1-infected donors, anti-CD40-CD40L fused to HIV-1 antigens preferentially expanded HIV-1-specific CD8+ T cells versus CD4+ T cells compared to analogous anti-CD40-antigen constructs. In normal donors, anti-CD40-CD40L-mediated delivery of Influenza M1 protein elicited M1-specific T cell expansion at lower doses compared to anti-CD40-mediated delivery. Also, on human myeloid-derived dendritic cells, anti-CD40-CD40L-melanoma gp100 peptide induced more sustained Class I antigen presentation compared to anti-CD40-gp100 peptide. In human CD40 transgenic mice, anti-CD40-CD40L-HIV-1 gp140 administered without adjuvant elicited superior antibody responses compared to anti-CD40-gp140 antigen without fused CD40L. In human CD40 mice, compared to the anti-CD40 vehicle, anti-CD40-CD40L delivery of Eα 52-68 peptide elicited proliferating of TCR I-Eα 52-68 CD4+ T cells producing cytokine IFNγ. Also, compared to controls, only anti-CD40-CD40L-Cyclin D1 vaccination of human CD40 mice reduced implanted EO771.LMB breast tumor cell growth. These data demonstrate that human CD40-CD40L antibody fused to antigens maintains highly agonistic activity and generates immune responses distinct from existing low agonist anti-CD40 targeting formats. These advantages were in vitro skewing responses towards CD8+ T cells, increased efficacy at low doses, and longevity of MHC Class I peptide display; and in mouse models, a more robust humoral response, more activated CD4+ T cells, and control of tumor growth. Thus, the anti-CD40-CD40L format offers an alternate DC-targeting platform with unique properties, including intrinsic adjuvant activity.

scholarly journals Pharmacological Modulation of SAMHD1 Activity by CDK4/6 Inhibitors Improves Anticancer Therapy

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 713 ◽  
Author(s):  
Marc Castellví ◽  
Eudald Felip ◽  
Ifeanyi Ezeonwumelu ◽  
Roger Badia ◽  
Edurne Garcia-Vidal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Anticancer Drug ◽  
Cell Cycle Progression ◽  
Drug Efficacy ◽  
Nucleotide Analogs ◽  
Central Process ◽  
Dntp Pool ◽  
Pharmacological Modulation ◽  
Intracellular Dntp ◽  
Samhd1 Expression

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 (CI < 0.04). 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.

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