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

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.

CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels.

In this study, we report that the tetraspanin CD81 enhances human immunodeficiency virus (HIV)-1 reverse transcription in HIV-1-infected cells. This is enabled by the direct interaction of CD81 with the deoxynucleoside triphosphate phosphohydrolase SAMHD1. This interaction prevents endosomal accumulation and favours the proteasome-dependent degradation of SAMHD1. Consequently, CD81 depletion results in SAMHD1 increased expression, decreasing the availability of deoxynucleoside triphosphates (dNTP) and thus HIV-1 reverse transcription. Conversely, CD81 overexpression, but not the expression of a CD81 carboxy (C)-terminal deletion mutant, increases cellular dNTP content and HIV-1 reverse transcription. Our results demonstrate that the interaction of CD81 with SAMHD1 controls the metabolic rate of HIV-1 replication by tuning the availability of building blocks for reverse transcription, namely dNTPs. Together with its role in HIV-1 entry and budding into host cells, the data herein indicate that HIV-1 uses CD81 as a rheostat that controls different stages of the infection.

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

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.

A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages.

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions.

HPV31 utilizes the ATR-Chk1 pathway to maintain elevated RRM2 levels and a replication-competent environment in differentiating Keratinocytes.

Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication.

Mechanistic and Kinetic Differences between Reverse Transcriptases of Vpx Coding and Non-coding Lentiviruses.

Among lentiviruses, HIV Type 2 (HIV-2) and many simian immunodeficiency virus (SIV) strains replicate rapidly in non-dividing macrophages, whereas HIV Type 1 (HIV-1) replication in this cell type is kinetically delayed. The efficient replication capability of HIV-2/SIV in non-dividing cells is induced by a unique, virally encoded accessory protein, Vpx, which proteasomally degrades the host antiviral restriction factor, SAM domain- and HD domain-containing protein 1 (SAMHD1). SAMHD1 is a dNTPase and kinetically suppresses the reverse transcription step of HIV-1 in macrophages by hydrolyzing and depleting cellular dNTPs. In contrast, Vpx, which is encoded by HIV-2/SIV, kinetically accelerates reverse transcription by counteracting SAMHD1 and then elevating cellular dNTP concentration in non-dividing cells. Here, we conducted the pre-steady-state kinetic analysis of reverse transcriptases (RTs) from two Vpx non-coding and two Vpx coding lentiviruses. At all three sites of the template tested, the two RTs of the Vpx non-coding viruses (HIV-1) displayed higher kpol values than the RTs of the Vpx coding HIV-2/SIV, whereas there was no significant difference in the Kd values of these two groups of RTs. When we employed viral RNA templates that induce RT pausing by their secondary structures, the HIV-1 RTs showed more efficient DNA synthesis through pause sites than the HIV-2/SIV RTs, particularly at low dNTP concentrations found in macrophages. This kinetic study suggests that RTs of the Vpx non-coding HIV-1 may have evolved to execute a faster kpol step, which includes the conformational changes and incorporation chemistry, to counteract the limited dNTP concentration found in non-dividing cells and still promote efficient viral reverse transcription.

Kinetic variations between reverse transcriptases of viral protein X coding and noncoding lentiviruses.

BACKGROUND: Host SAM domain and HD domain-containing protein 1 (SAMHD1) suppresses reverse transcription kinetics of HIV-1 in nondividing cells such as macrophages by hydrolyzing and nearly depleting cellular dNTPs, which are the substrates of viral reverse transcriptase (RT). However, unlike HIV-1, HIV-2 and SIVsm encode viral protein X (Vpx), which counteracts the dNTPase activity of SAMHD1 and elevates dNTP concentration, allowing the viruses to replicate under abundant dNTP conditions even in nondividing cells. FINDINGS: Here we tested whether RTs of these Vpx coding and noncoding lentiviruses display different enzyme kinetic profiles in response to dNTP concentrations. For this test, we characterized an extensive collection of RTs from 7 HIV-1 strains, 4 HIV-2 strains and 7 SIV strains, and determined their steady-state kinetic parameters. The K m values of all HIV-1 RTs were consistently low and close to the low dNTP concentrations found in macrophages. However, the K m values of SIV and HIV-2 RTs were not only higher than those of HIV-1 RTs but also varied significantly, indicating that HIV-2/SIV RTs require higher dNTP concentrations for efficient DNA synthesis, compared to HIV-1 RT. However, the k cat values of all eighteen lentiviral RTs were very similar. CONCLUSIONS: Our biochemical analysis supports the hypothesis that the enzymological properties, particularly, K m values, of lentivirus RTs, are mechanistically tied with the cellular dNTP availability in nondividing target cells, which is controlled by SAMHD1 and Vpx.

dNTP pool modulation dynamics by SAMHD1 protein in monocyte-derived macrophages.

BACKGROUND: SAMHD1 degrades deoxyribonucleotides (dNTPs), suppressing viral DNA synthesis in macrophages. Recently, viral protein X (Vpx) of HIV-2/SIVsm was shown to target SAMHD1 for proteosomal degradation and led to elevation of dNTP levels, which in turn accelerated proviral DNA synthesis of lentiviruses in macrophages. RESULTS: We investigated both time-dependent and quantitative interplays between SAMHD1 level and dNTP concentrations during multiple exposures of Vpx in macrophages. The following were observed. First, SAMHD1 level was rapidly reduced by Vpx + VLP to undetectable levels by Western blot analysis. Recovery of SAMHD1 was very slow with less than 3% of the normal macrophage level detected at day 6 post Vpx treatment and only ~30% recovered at day 14. Second, dGTP, dCTP and dTTP levels peaked at day 1 post Vpx treatment, whereas dATP peaked at day 2. However, all dNTPs rapidly decreased starting at day 3, while SAMHD1 level was below the level of detection. Third, when Vpx pretreated macrophages were re-exposed to a second Vpx treatment at day 7, we observed dNTP elevation that had faster kinetics than the first Vpx + VLP treatment. Moreover, we performed a short kinetic analysis of the second Vpx treatment to find that dATP and dGTP levels peaked at 8 hours post secondary VLP treatment. dGTP peak was consistently higher than the primary, whereas peak dATP concentration was basically equivalent to the first Vpx + VLP treatment. Lastly, HIV-1 replication kinetics were faster in macrophages treated after the secondary Vpx treatments when compared to the initial single Vpx treatment. CONCLUSION: This study reveals that a very low level of SAMHD1 sufficiently modulates the normally low dNTP levels in macrophages and proposes potential diverse mechanisms of Vpx-mediated dNTP regulation in macrophages.

Vesicular integrity in Parkinson's disease.

The defining motor characteristics of Parkinson's disease (PD) are mediated by the neurotransmitter dopamine (DA). Dopamine molecules spend most of their lifespan stored in intracellular vesicles awaiting release and very little time in the extracellular space or the cytosol. Without proper packaging of transmitter and trafficking of vesicles to the active zone, dopamine neurotransmission cannot occur. In the cytosol, dopamine is readily oxidized; excessive cytosolic dopamine oxidation may be pathogenic to nigral neurons in PD. Thus, factors that disrupt vesicular function may impair signaling and increase the vulnerability of dopamine neurons. This review outlines the many mechanisms by which disruption of vesicular function may contribute to the pathogenesis of PD. From direct inhibition of dopamine transport into vesicles by pharmacological or toxicological agents to alterations in vesicle trafficking by PD-related gene products, variations in the proper compartmentalization of dopamine can wreak havoc on a functional dopamine pathway. Findings from patient populations, imaging studies, transgenic models, and mechanistic studies will be presented to document the relationship between impaired vesicular function and vulnerability of the nigrostriatal dopamine system. Given the deleterious effects of impaired vesicular function, strategies aimed at enhancing vesicular function may be beneficial in the treatment of PD.