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

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.

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

“…In vitro, quiescent CD4 ϩ T cells, myeloid cells (including macrophages and dendritic cells), and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells, express relatively high levels of SAMHD1 and restrict HIV-1 replication, likely because the deoxynucleoside triphosphohydrolase (dNTPase) activity of SAMHD1 leads to ablation of viral reverse transcription (14,(16)(17)(18)(19)(20). However, proliferating cells are highly permissive for infection and replication of HIV-1 (21)(22)(23). Together with upregulation of ribonucleotide reductase (RNR), downregulation of SAMHD1 expression and dNTPase activity during the S phase was considered a characteristic feature of proliferating cells (24,25) until recently; Schott et al (in 2018) and Yan et al (in 2015) reported that SAMHD1 levels remain relatively unchanged during the cell cycle (26,27).…”

“…In cycling cells, p21 can be targeted for proteasomal degradation by the S-phase-associated protein 2 (SKP2) in a complex with cyclin A/E and CDK2 (36,37). p21 blocks dNTP biosynthesis in monocyte-derived macrophages and dendritic cells by downmodulating the expression of the RNR2 subunit of ribonucleotide reductase, which is essential for the reduction of ribonucleotides to deoxynucleotides (23,25,38). On the other hand, the ability of p21 to inhibit cyclin/CDK activities likely regulates SAMHD1 antiviral activity by prohibiting the cyclinA/CDK1/2 phosphorylation of SAMHD1 at T592 (21,22,30,39), suggesting that p21 regulates both de novo dNTP synthesis and the antiviral function of SAMHD1 (23,39,40).…”

Anti-apoptotic clone 11 derived peptides induce in vitro death of CD4+ T cells susceptible to HIV-1 infection

“…Although further studies will be necessary to directly evaluate this, our results suggest that HIV-1 may rely on AAC-11-dependent anti-apoptotic pathway for the establishment of productive infection in CD4+ T cells. AAC-11 gene expression, besides strongly correlating with multiple genes involved in the regulation of cell metabolism, was also correlated with the expression of RRM2 (Fig 1C), an enzyme that is critical for the de novo synthesis of dNTPs, and the depletion of which blocks HIV-1 infection in macrophages and dendritic cells (44, 45). AAC-11 mRNA levels also correlated with the expression of other genes that have been associated with the HIV replication cycle, in particular trafficking (i.e.…”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…IFN1 signaling impedes cell cycle progression by up-regulating the cell cycle inhibitor p21 cip1/waf1 which subsequently restricts the activity of CDKs (123). Accordingly, monocyte derived dendritic cells (MDDC) matured in the presence of IFN-γ and CD40L, or M-CSF, demonstrated a decrease in phosphorylated SAMHD1 that corresponded to an increase in p21 cip1/waf and a decrease in cellular dNTP pools (124, 125). DNA damage can also activate p21 and result in the concomitant loss of phosphorylated SAMHD1 in a p53 dependent manner, as described in a recent study in which macrophages were treated with topoisomerase inhibitors in order to induce a DNA damage response (126).…”

“…The loss of phosphorylation at T592 coincides with the reemergence of a cellular phenotype that is refractory to HIV-1 infection and provides a clear linkage between SAMHD1 and innate immunity, while emphasizing the importance of SAMHD1 phosphorylation status as a determinant of viral restriction. While phosphorylation of T592 has been established as coinciding with the ablation of the viral restriction capacity, some data suggest that this loss of antiviral capacity is not entirely coupled to diminished SAMHD1 catalytic activity (116, 117, 125, 169). Cells expressing phosphomimetic T592D or T592E mutations support this model, as they are unable to restrict viral infection but do not exhibit elevated dNTP pools or reduced dNTPase activity (81, 116, 169).…”