Suresh de Silva

SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+T-lymphocytes cannot be upregulated by interferons

BackgroundSAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs), macrophages, and resting CD4+ T-cells. Acting as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4+ T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN)-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4+ T-lymphocytes is unknown.ResultsHere, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4+ T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool.ConclusionsOur results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not upregulated by type I IFNs.

Multimode, Cooperative Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors

Background: 2-(Quinolin-3-yl)-acetic-acid derivatives target HIV-1 integrase and inhibit viral replication. Results: The compounds are allosteric integrase inhibitors (ALLINIs) that block integrase interactions with viral DNA and its cellular cofactor LEDGF and cooperatively inhibit HIV-1 replication. Conclusion: ALLINIs block multiple steps of HIV-1 integration. Significance: These new properties of ALLINIs will facilitate their further development as potent antiretroviral compounds. The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC50 values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.

Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

ABSTRACT Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4+ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4+ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. IMPORTANCE SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.

Co-Evolution of Primate SAMHD1 and Lentivirus Vpx Leads to the Loss of the vpx Gene in HIV-1 Ancestor

Cross-species transmission and adaptation of simian immunodeficiency viruses (SIVs) to humans have given rise to human immunodeficiency viruses (HIVs). HIV type 1 (HIV-1) and type 2 (HIV-2) were derived from SIVs that infected chimpanzee (SIVcpz) and sooty mangabey (SIVsm), respectively. The HIV-1 restriction factor SAMHD1 inhibits HIV-1 infection in human myeloid cells and can be counteracted by the Vpx protein of HIV-2 and the SIVsm lineage. However, HIV-1 and its ancestor SIVcpz do not encode a Vpx protein and HIV-1 has not evolved a mechanism to overcome SAMHD1-mediated restriction. Here we show that the co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in SIVcpz and HIV-1. We found evidence for positive selection of SAMHD1 in orangutan, gibbon, rhesus macaque, and marmoset, but not in human, chimpanzee and gorilla that are natural hosts of Vpx-negative HIV-1, SIVcpz and SIVgor, respectively, indicating that vpx drives the evolution of primate SAMHD1. Ancestral host state reconstruction and temporal dynamic analyses suggest that the most recent common ancestor of SIVrcm, SIVmnd, SIVcpz, SIVgor and HIV-1 was a SIV that had a vpx gene; however, the vpx gene of SIVcpz was lost approximately 3643 to 2969 years ago during the infection of chimpanzees. Thus, HIV-1 could not inherit the lost vpx gene from its ancestor SIVcpz. The lack of Vpx in HIV-1 results in restricted infection in myeloid cells that are important for antiviral immunity, which could contribute to the AIDS pandemic by escaping the immune responses.

Promoter Methylation Regulates SAMHD1 Gene Expression in Human CD4+ T Cells

Background: SAMHD1 expression is significantly reduced in CD4+ T cell lines compared with primary CD4+ T lymphocytes. Results: The SAMHD1 promoter contains a CpG island that is prone to DNA methylation. Conclusion: Methylation of the SAMHD1 promoter contributes to transcriptional repression in CD4+ T cell lines. Significance: We identified promoter methylation as a contributing factor to SAMHD1 gene regulation. The retrovirus restriction factor SAMHD1 is the first identified mammalian dNTP triphosphohydrolase that is highly expressed in human myeloid lineage cells and CD4+ T lymphocytes. Although SAMHD1 expression is variable in human cell lines and tissue types, mechanisms underlying SAMHD1 gene regulation have not been defined. Recent studies showed that SAMHD1 is highly expressed in human primary CD4+ T lymphocytes, but not in some CD4+ T cell lines. Here, we report that SAMHD1 expression varies among four CD4+ T cell lines and is transcriptionally regulated. Cloning and sequence analysis of the human SAMHD1 promoter revealed a CpG island that is methylated in CD4+ T cell lines (such as Jurkat and Sup-T1), resulting in transcriptional repression of SAMHD1. We also found that the SAMHD1 promoter is unmethylated in primary CD4+ T lymphocytes, which express high levels of SAMHD1, indicating a direct correlation between the methylation of the SAMHD1 promoter and transcriptional repression. SAMHD1 expression was induced in CD4+ T cell lines by blocking DNA methyltransferase activity, suggesting that promoter methylation is one of the key epigenetic mechanisms by which SAMHD1 expression is regulated.

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

To the Editor: Sterile alpha motif domain– and HD domain–containing protein 1 (SAMHD1) is a cellular dNTP triphosphohydrolase (dNTPase) that restricts HIV-1 replication in myeloid cells and resting CD4+ T cells by degrading dNTPs and limiting viral reverse transcription1–5. Purified recombinant SAMHD1 also has exonuclease activity when synthetic nucleic acids or HIV-1 gag and tat RNAs transcribed in vitro are used as substrates6. Ryoo et al.7 recently suggested that SAMHD1 restricts HIV-1 infection through its ribonuclease (RNase) activity by cleaving the viral RNA genome. By using SAMHD1 mutants purported to specifically retain dNTPase (SAMHD1Q548A) or RNase (SAMHD1D137N) activities, Ryoo et al.7,8 proposed that the RNase activity of SAMHD1, but not its dNTPase activity, is essential for HIV-1 restriction in nondividing cells. They also suggested that SAMHD1 phosphorylation at T592 negatively regulated its RNase activity7. To extend these findings7, we measured HIV-1 protein synthesis and virion production in the presence of SAMHD1 when the requirement for intracellular dNTP-dependent HIV-1 reverse transcription was bypassed. We co-transfected an HIV-1 proviral DNA plasmid (pNL4-3) with a plasmid expressing wild-type (WT) SAMHD1 or a phosphoablative, but dNTPase-active, mutant (SAMHD1T592A; refs. 9–11) into human embryonic kidney (HEK) 293T cells and assessed intracellular HIV-1 Gag protein synthesis and viral particle release in the supernatants. This transfection-based HIV-1 production is independent of reverse transcription requiring intracellular dNTPs as precursors of viral DNA synthesis, but is dependent on HIV-1 mRNA–mediated gene expression. Intracellular HIV-1 Gag protein levels, p24 capsid levels in released HIV-1 virions and infectivity were not reduced by the ectopic expression of WT SAMHD1 or SAMHD1T592A mutant (Supplementary Fig. 1), which suggests that SAMHD1 cannot inhibit HIV-1 production after the reverse transcription step, regardless of its phosphorylation at T592. These findings are consistent with our previous results showing that the expression of WT SAMHD1 or of the SAMHD1T592A mutant in dividing cells does not restrict HIV-1 infection11,12. Our results suggest that SAMHD1 does not have broad nuclease activity, but do not rule out a specific nucleolytic interaction between SAMHD1 and incoming HIV-1 genomic RNA (gRNA). Given the preponderance of previous data implicating the dNTPase activity of SAMHD1 as its primary antiviral mechanism, we reproduced the key experiments of Ryoo et al.7. Their conclusion that SAMHD1 restricts HIV-1 through RNase activity was based on the differential activity of the SAMHD1 mutants, SAMHD1D137N and SAMHD1Q548A (ref. 7). We independently generated these two mutant constructs and confirmed the expected mutations by DNA sequencing to ensure that there were no other disabling mutations in the constructs. We then examined the HIV-1 restriction and intracellular dNTP regulation by these mutants and WT SAMHD1 by following the protocol of Ryoo et al.7. Our results show that both the SAMHD1D137N and SAMHD1Q548A mutants were expressed at similar levels to that of WT SAMHD1, and each efficiently restricted HIV-1 infection and decreased dATP, dGTP and dTTP levels in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells (Fig. 1a–c). SAMHD1 expression did not significantly decrease dCTP levels, as compared to vector control cells (Fig. 1c, right), probably owing to the different biosynthesis pathway of dCTP. Notably, Ryoo et al.7 showed only dCTP levels, but not dATP, dGTP or dTTP levels, in SAMHD1expressing or control cells. Previous studies have used a SAMHD1D137A mutant to explore the effects of the dGTP binding site (D137) on the dNTPase activity, tetramer formation and HIV-1 restriction of SAMHD1 (refs. 13–15). The SAMHD1D137A mutant has no detectable dNTPase activity or HIV-1 restriction in vitro, owing to its inability to form a stable tetramer14,15. It is possible that the SAMHD1D137N mutant might be stabilized in cells to remain in a tetrameric form, thus maintaining its ability to reduce intracellular dNTP levels and restrict HIV-1 infection. It is also possible that an in vitro dNTPase assay using purified recombinant SAMHD1 proteins might not fully reflect the dNTPase activity of SAMHD1 in PMA-differentiated U937 cells. However, these possibilities remain to be examined to explain how the SAMHD1D137N mutant restricts HIV-1 infection if its dNTPase activity is impaired. In contrast to the results of Ryoo et al.7, the SAMHD1D137N and SAMHD1Q548A mutants in our experiments do not have differing anti-HIV-1 activities, and neither lacks the ability to lower cellular dNTP levels. Ryoo et al.7 reported an approximately twofold decrease in HIV-1 gRNA levels in PMA-differentiated U937 cells expressing WT SAMHD1 or SAMHD1D137N, but not the SAMHD1Q548A mutant, as compared to the control cells at 3 h and 6 h postinfection (h.p.i.), which suggests SAMHD1-mediated HIV-1 gRNA degradation7. By contrast, we detected comparable levels of HIV-1 gRNA in PMAdifferentiated U937 cells expressing SAMHD1 (WT, SAMHD1D137N and SAMHD1Q548A mutants) and the vector control cells at 1, 3 and 6 h.p.i., respectively (Fig. 1d), showing that, in our study, SAMHD1 cannot degrade HIV-1 gRNA during early infection. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in infected cells at 12 and 24 h.p.i, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. We found that the expression of WT SAMHD1, SAMHD1D137N and SAMHD1Q548A mutants significantly reduced HIV-1 late reverse transcription products as compared to the vector control cells (Fig. 1e), correlating well with a reduced intracellular dNTP pool (Fig. 1c). Thus, in our study, these two mutants of SAMHD1 cannot distinguish its dNTPase and RNase functions, and dNTP depletion accounts for SAMHD1-mediated HIV-1 restriction. Seamon et al.16 reported that trace exonuclease activities of recombiSAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

Differential Effects of Vpr on Single-cycle and Spreading HIV-1 Infections in CD4+ T-cells and Dendritic Cells

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) contributes to viral replication in non-dividing cells, specifically those of the myeloid lineage. However, the effects of Vpr in enhancing HIV-1 infection in dendritic cells have not been extensively investigated. Here, we evaluated the role of Vpr during infection of highly permissive peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells and compared it to that of monocyte-derived dendritic cells (MDDCs), which are less susceptible to HIV-1 infection. Infections of dividing PBMCs and non-dividing MDDCs were carried out with single-cycle and replication-competent HIV-1 encoding intact Vpr or Vpr-defective mutants. In contrast to previous findings, we observed that single-cycle HIV-1 infection of both PBMCs and MDDCs was significantly enhanced in the presence of Vpr when the viral stocks were carefully characterized and titrated. HIV-1 DNA quantification revealed that Vpr only enhanced the reverse transcription and nuclear import processes in single-cycle HIV-1 infected MDDCs, but not in CD4+ T-cells. However, a significant enhancement in HIV-1 gag mRNA expression was observed in both CD4+ T-cells and MDDCs in the presence of Vpr. Furthermore, Vpr complementation into HIV-1 virions did not affect single-cycle viral infection of MDDCs, suggesting that newly synthesized Vpr plays a significant role to facilitate single-cycle HIV-1 infection. Over the course of a spreading infection, Vpr significantly enhanced replication-competent HIV-1 infection in MDDCs, while it modestly promoted viral infection in activated PBMCs. Quantification of viral DNA in replication-competent HIV-1 infected PBMCs and MDDCs revealed similar levels of reverse transcription products, but increased nuclear import in the presence of Vpr independent of the cell types. Taken together, our results suggest that Vpr has differential effects on single-cycle and spreading HIV-1 infections, which are dependent on the permissiveness of the target cell.

Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection

Human SAMHD1 (hSAMHD1) restricts HIV-1 infection in non-dividing cells by depleting intracellular dNTPs to limit viral reverse transcription. Phosphorylation of hSAMHD1 at threonine (T) 592 by cyclin-dependent kinase (CDK) 1 and CDK2 negatively regulates HIV-1 restriction. Mouse SAMHD1 (mSAMHD1) restricts HIV-1 infection in non-dividing cells, but whether its phosphorylation regulates retroviral restriction is unknown. Here we identified six phospho-sites of mSAMHD1, including T634 that is homologous to T592 of hSAMHD1 and phosphorylated by CDK1 and CDK2. We found that wild-type (WT) mSAMHD1 and a phospho-ablative mutant, but not a phospho-mimetic mutant, restricted HIV-1 infection in differentiated U937 cells. Murine leukemia virus (MLV) infection of dividing NIH3T3 cells was modestly restricted by mSAMHD1 WT and phospho-mutants, but not by a dNTPase-defective mutant. Our results suggest that phosphorylation of mSAMHD1 at T634 by CDK1/2 negatively regulates its HIV-1 restriction in differentiated cells, but does not affect its MLV restriction in dividing cells.

TRIM5 Acts as More Than a Retroviral Restriction Factor

The retrovirus restriction factor TRIM5α blocks post-entry infection of retroviruses in a species-specific manner. As a cellular E3 ubiquitin ligase, TRIM5α binds to the retroviral capsid lattice in the cytoplasm of an infected cell and accelerates the uncoating process of retroviral capsid, thus providing a potent restriction to HIV-1 and other retrovirus infections. The precise mechanism by which this restriction is imposed remains under scrutiny, and evidence is lacking to link the E3 ubiquitin ligase activity of TRIM5α to its ability to restrict retrovirus infection. In a recent study, Pertel and colleagues have uncovered the link between the two, providing compelling evidence to suggest that following the interaction with the retroviral capsid, TRIM5 triggers an antiviral innate immune response by functioning as a pattern recognition receptor [1]. This unique function of TRIM5 is dependent on its association with the E2 ubiquitin-conjugating enzyme complex UBC13-UEV1A and subsequent activation of the TAK1 kinase complex and downstream genes involved in innate immune responses. These findings have defined a novel function for TRIM5 as a pattern recognition receptor in innate immune recognition and provided valuable mechanistic insight into its role as a retroviral restriction factor. Here we discuss the significance of these new findings in understanding TRIM5-mediated HIV restriction.

Cutaneous T-Cell Lymphoma: Mycosis Fungoides and Sézary Syndrome

Mycosis fungoides (MF) and Sezary syndrome (SS) are the most common variants of cutaneous T-cell lymphomas (CTCLs) and generally have an indolent course. These are subtypes of a diverse group of lymphoid malignancies that develop in the skin, which includes both indolent and aggressive types. MF/SS are neoplasms of CD4+ T cells, expressing a memory effector phenotype. In the early stages, MF/SS can be challenging to differentiate from nonmalignant inflammatory skin disorders, such as psoriasis or nonspecific dermatitis and additional specific biomarkers would improve diagnosis. Gene expression analysis has uncovered many novel transcriptional abnormalities in MF/SS, hinting at epigenetic mechanisms potentially playing a role in the altered regulation. The mechanism for the epigenetic changes is unclear, but these abnormal genes may serve as biomarkers in MF/SS diagnosis, provide insight into the pathogenesis of MF/SS, and lead to new treatment approaches.