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HIV Protein called SAMHD1: New HIV Research Advance Reveals How Protein Protects Cells from HIV Infection - Potential New Drug Therapy
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A quantitative basis for antiretroviral therapy for HIV-1 infection by R Siliciano et al - (02/23/12)
from study authors:
"By depleting the pool of available dNTPs, SAMHD1 effectively starves the virus of a building block that is central to its replication strategy. The use of Vpx to destroy SAMHD1 represents a notable strategy developed by lentiviruses to thwart cellular antiviral defenses. These findings bring to light a previously unknown mechanism of innate immunity and raise the question of whether the pharmacological alteration of intracellular dNTP pools might be a therapeutic approach to treating virus infection. The implications of depleting the nucleotide pool as a host-defense mechanism are potentially far-reaching."
New research reveals how protein protects cells from HIV infection
Finding offers potential new drug targets aimed at slowing progression of disease
NEW YORK 12-Feb-2012 -- A novel discovery by researchers at NYU Langone Medical Center and colleagues reveals a mechanism by which the immune system tries to halt the spread of HIV. Harnessing this mechanism may open up new paths for therapeutic research aimed at slowing the virus' progression to AIDS. The study appears online ahead of print today in Nature Immunology.
"A lot of research on viruses, especially HIV, is aimed at trying to understand what the body's mechanisms of resistance are and then to understand how the virus has gotten around these mechanisms," said co-lead investigator Nathaniel R. Landau, PhD, a professor of microbiology at the Joan and Joel Smilow Research Center at NYU School of Medicine.
The research focused on a protein called SAMHD1. Recent studies have found that immune cells, called dendritic cells, containing the protein are resistant to infection by HIV. Since the discovery, scientists have sought to understand how SAMHD1 works to protect these cells, with hopes that science might find a way to synthetically apply that protection to other cells.
Dr. Landau and his team are now able to provide an answer:
When a virus, like HIV, infects a cell, it hijacks the cell's molecular material to replicate. That molecular material is in the form of deoxynucleotide triphosphates (dNTPs), which are the building blocks for DNA. Once the virus replicates, the resulting DNA molecule contains all the genes of the virus and instructs the cell to make more virus.
Researchers wanted to understand how cells containing the SAMHD1 protein are protected from such hijacking. They found that SAMHD1 protects the cell from viruses by destroying the pool of dNTPs, leaving the virus without any building blocks to make its genetic information - a process researchers call nucleotide pool depletion. "SAMHD1 essentially starves the virus," Dr. Landau said. "The virus enters the cell and then nothing happens. It has nothing to build and replicate with, so no DNA is made."
As a result, the most common form of HIV does not readily infect these cells. Instead, the virus has evolved to replicate mainly in a different kind of cell, called CD4 T-cells, which do not contain SAMHD1 and therefore have a healthy pool of dNTPs. Dr. Landau explained that the virus has evolved in such a way that it may deliberately avoid trying to infect immune cells with SAMHD1 to avoid alerting the greater immune system to activate a variety of antiviral mechanisms to attack the virus. Viruses that are related to HIV, like HIV-2 and SIV, have developed a protein called viral protein X (VPX) that directly attacks SAMHD1. This allows the virus to infect dendritic cells, an important type of immune cell.
"Viruses are remarkably clever about evading our immune defenses," Dr. Landau said. "They can evolve quickly and have developed ways to get around the systems we naturally have in place to protect us. It's a bit of evolutionary warfare and the viruses, unfortunately, usually win. We want to understand how the enemy fights so that we can outsmart it in the end."
Understanding the mechanism by which SAMHD1 provides protection to cells may provide a new idea about how to stop or slow the virus' ability to spread, Dr. Landau explained. Potential future research efforts, for example, might focus on finding a way to increase the amount of SAMHD1 in cells where it does not exist, or to reduce the amount of dNTPs in cells vulnerable to infection.
"Over the past few years, a number of these natural resistance mechanisms have been identified, specifically in HIV, but some have potential applications to other viruses, as well," he said. "This is a very exciting time in HIV research. Many of the virus' secrets are being revealed through molecular biology, and we're learning a tremendous amount about how our immune system works through the study of HIV."
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Funded in part by the National Institutes of Health and the American Foundation for AIDS Research, the study was conducted in collaboration with researchers at several institutions, including the University of Rochester Medical Center and The Cochin Institute, in Paris.
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Nature Immunology | Article March issue 2012
"By depleting the pool of available dNTPs, SAMHD1 effectively starves the virus of a building block that is central to its replication strategy. The use of Vpx to destroy SAMHD1 represents a notable strategy developed by lentiviruses to thwart cellular antiviral defenses. These findings bring to light a previously unknown mechanism of innate immunity and raise the question of whether the pharmacological alteration of intracellular dNTP pools might be a therapeutic approach to treating virus infection. The implications of depleting the nucleotide pool as a host-defense mechanism are potentially far-reaching."
SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates - pdf attached
Abstract
SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.
Introduction
Myeloid cells such as dendritic cells (DCs) and monocyte-derived macrophages (MDMs) have an important role in orchestrating the innate and adaptive immune responses to virus infection. They also serve as natural targets of lentiviruses, including human immunodeficiency virus type 1 (HIV-1), that efficiently transmit the virus to activated CD4+ T cells and serve as a long-term reservoir that sustains ongoing virus infection. Infection of MDMs and DCs by HIV-1 is inefficient, which suggests that these cells may express a myeloid-specific restriction factor. Viruses of the HIV-2-simian immunodeficiency virus of sooty mangabeys (SIVsm) lineage express the accessory protein Vpx, which enhances their ability to infect myeloid cells and has been proposed to counteract the putative restriction factor1. The MDM- and DC-expressed target of Vpx has been identified as SAMHD1, a protein that contains a sterile α-motif and an HD domain2, 3.
The gene encoding human SAMHD1 was initially identified as an ortholog of a mouse gene induced by interferon-γ and in response to viral infection4. SAMHD1 has been proposed to act as a negative regulator of the innate immune response, analogous to TREX-1, a cytoplasmic exonuclease of single-stranded DNA that is thought to prevent the accumulation of virus and retrotransposon DNA to dampen innate immune responses5. Mutations in the genes encoding SAMHD1, TREX1 and RNase H2 are associated with Aicardi-Goutieres syndrome, a rare early-onset inflammatory encephalopathy characterized by inappropriate immune activation and overproduction of interferon-α6, 7. The SAMHD1 protein consists of an amino-terminal sterile α-motif domain and a central HD domain with putative nucleotidase and phosphodiesterase activities. Point mutations of the sequence encoding the conserved catalytic amino acid residues in the SAMHD1 HD domain inactivate its lentivirus-restriction activity2, 3, 6, 8. SAMHD1 has weak homology with EF1143, an Enterococcus fecalis HD domain-containing bacterial nucleotide-metabolic enzyme9, which further suggests that SAMHD1 has a role in nucleotide biochemistry. Homozygous deletions in the gene encoding SAMHD1 are associated with deletions of mitochondrial DNA, and mitochondrial diseases are often the result of mutations in genes that encode enzymes involved in the metabolism of deoxynucleoside triphosphates (dNTPs)10, 11, 12. The association of HD domains with phosphohydrolase activity8, 9 suggests that the antiviral activity of SAMHD1 could be mediated by an effect on dNTP metabolism. DCs have high expression of SAMHD1; MDMs have moderate SAMHD1 expression; and HIV-1-permissive CD4+ T cells have only low SAMHD1 expression3.
Cells differ in their dNTP content depending on cell type, differentiation and activation state and cell cycle13. The dNTP concentration in terminally differentiated, nonreplicating MDMs (20-40 nM) is less than 1% that of activated CD4+ T cells (2-4 μM). Lentiviruses have evolved to replicate under conditions of low dNTP concentrations by virtue of a reverse transcriptase (RT) with a low Michaelis constant (Km) for dNTPs13, 14. Nevertheless, reverse transcription of HIV-1 proceeds more slowly in MDMs than in activated T cells15, 16. The addition of deoxynucleosides to the culture medium accelerates the reverse transcription of HIV-1 in infected MDMs, which indicates that the intracellular dNTP pool is limiting in these cells13, 14, 16, 17. Despite having an efficient RT, viruses of the HIV-2-SIVsm lineage depend on Vpx for the productive infection of monocytic cells18, 19.
Viruses of the HIV-2-SIVsm lineage express the accessory protein Vpx, which counteracts SAMHD1-mediated restriction2, 3. Vpx is a small nuclear protein that is packaged in virions through a specific interaction with an amino acid motif in the p6 protein of the major virion structural precursor polyprotein Gag20, 21. In the host cell, Vpx is associated with the multisubunit cullin 4A-ring E3 ubiquitin ligase CRL4. When expressed in a cell or introduced exogenously via virus-like particles (VLPs), Vpx binds to SAMHD1, and the associated CRL4 complex induces the ubiquitination and subsequent degradation of SAMHD1. HIV-1 does not express Vpx yet is highly sensitive to SAMHD1-mediated restriction21, 22.
Here we demonstrate that SAMHD1 lowered the concentration of intracellular dNTPs in myeloid cells to an amount that failed to support reverse transcription and thereby established a cellular state that was not permissive to lentiviral infection. Vpx induced degradation of SAMHD1, which resulted in a larger intracellular dNTP pool and restored permissiveness of the cell to infection. Although SIVmac Vpx counteracted human and rhesus SAMHD1, it was not active against the mouse homolog of SAMHD1.
Discussion
Our findings have suggested that the main mechanism by which SAMHD1 restricts lentivirus infection of myeloid cells is by decreasing the concentration of dNTPs to below the Km of RT, thereby blocking reverse transcription. It has been reported that recombinant SAMHD1 is a dNTP triphosphohydrolase30, 31, a finding that we have confirmed here and that suggests SAMHD1 directly regulates the intracellular dNTP pool. HIV-2-SIVsm lentiviruses counteract SAMHD1 via Vpx brought into the target cell by the incoming virion. Vpx induced the proteosomal degradation of SAMHD1 in the target cells, which resulted in higher intracellular dNTP concentrations through new synthesis.
SAMHD1-mediated depletion of the intracellular dNTP pool provides a means by which the cell establishes an antiviral state without the need for an inhibitor that interacts directly with a specific viral component. Thus, this mechanism may serve to restrict a diverse range of retroviruses. In addition, this mechanism explains how SAMHD1 can block reverse transcription, which occurs in the cytoplasm, even though it is localized to the nucleus, a spatially separated cellular compartment. Furthermore, it provides a rationale for why SAMHD1 restriction operates only in terminally differentiated and nondividing cells such as MDMs and DCs. Those cells, in contrast to dividing cells, do not need to maintain a high concentration of dNTPs and therefore are not harmed by the catalytic activity of SAMHD1. Expression of a transduced gene encoding SAMHD1 in actively dividing cells had little effect on dNTP concentrations and did not restrict infection. Moreover, we detected endogenous SAMHD1 expression in activated CD4+ T cells, yet these cells maintained a high concentration of dNTPs (data not shown). These findings suggest that either the catalytic activity of the enzyme is regulated in actively replicating cells or the rate of dNTP production is sufficient to replenish the dNTP pool. Consistent with the latter possibility, treatment of cells with hydroxyurea to block dNTP synthesis demonstrated the ability of SAMHD1 to restrict infection. It is notable that Vpx packaged in the incoming virion was able to induce enough degradation of SAMHD1 to substantially increase the dNTP concentration in the target cell. SAMHD1 degradation has been demonstrated before through the use of Vpx-containing VLPs, but here we found that it also occurred when Vpx was packaged in the incoming virion.
HIV-1 is highly sensitive to SAMHD1-mediated restriction yet lacks Vpx. It is possible that in vivo, HIV-1 replication is sustained mainly by T cells and thus there is no selective advantage in maintaining the open reading frame of the gene encoding Vpx. Alternatively, SAMHD1 could provide a selective advantage to HIV-1 by limiting the ability of the virus to infect myeloid cells. These cells contain sensors for cytoplasmic DNA as well as a cryptic sensor that detects newly synthesized viral proteins32. It has been suggested that the lack of Vpx serves as a strategy by which the virus avoids activating cytoplasmic sensors and thereby limits the induction of inflammatory cytokines32. The identity of the viral protein sensor is not known and is probably not SAMHD1 itself. Nevertheless, SAMHD1 is interferon inducible33, and the cryptic sensor could serve to induce SAMHD1 expression in nearby cells.
The role of the Vpx-related lentiviral accessory protein Vpr in vivo remains poorly understood. Like Vpx, Vpr is packaged in virions and interacts with the E3 ubiquitin ligase CRL4 (refs. 34,35) but has only a modest effect on MDM and DC infection. Although SIVmac lacking Vpr or Vpx is only slightly less pathogenic than wild-type SIVmac in rhesus macaques, SIVmac lacking both Vpx and Vpr is highly attenuated, which suggests an overlap in function36. Unlike Vpx, SIVmac Vpr did not interact detectably with SAMHD1 and did not affect the concentration of dNTPs in cultured MDMs (data not shown). Nevertheless, it remains possible that Vpr influences a nucleotide-metabolic pathway that is not active under standard culture conditions and that therefore its effect is not detected. It is also possible that for some SIV isolates, Vpr interacts with SAMHD1 and has Vpx-like function.
In addition to its role in counteracting SAMHD1, Vpx is reported to bind to the cytidine deaminase APOBEC3A37, and it has been suggested that Vpx has a dual role in counteracting the two virus-restriction factors. This could be explained if the higher concentration of dNTPs that results from Vpx-induced degradation of SAMHD1 allows more rapid reverse transcription. The higher rate of viral DNA synthesis could then protect the transcript from deamination by APOBEC3A.
To replicate their genomes, DNA viruses and viruses that replicate through a DNA intermediate need to access dNTPs and have evolved various strategies to ensure their availability. Small DNA viruses 'preferentially' infect mitotic cells; adenoviruses, polyomaviruses and papillomaviruses encode proteins that drive quiescent cells into S phase38; and herpesviruses and poxviruses encode an ribonucleotide reductase that converts NTPs to dNTPs38, 39. For retroviruses, the limited supply of dNTPs in nondividing and quiescent cells presents a particular challenge24, 40. The requirement for high concentrations of dNTPs by gamma retroviruses impairs the reverse transcription of these viruses in quiescent and nondividing cells41, 42. Lentiviruses have adapted to this problem in part by developing an RT with a low Km for dNTP13, 14, 16, 17. Despite their efficient RT, HIV-1 and HIV-2 remain sensitive to the low concentration of dNTPs in the cytoplasm of their natural target cells. HIV-1 seemed to be more sensitive to SAMHD1-mediated restriction than was ∼vpx SIV and was more sensitive to the addition of deoxynucleosides to the culture medium (data not shown). HIV-1 in which RT was altered to decrease its affinity for dNTP was more sensitive to SAMHD1. These findings suggest that HIV-1 isolates may vary in their sensitivity to SAMHD1 and that variability in intracellular dNTP concentrations as a result of cellular activation state and genetic polymorphism could influence HIV-1 replication.
By depleting the pool of available dNTPs, SAMHD1 effectively starves the virus of a building block that is central to its replication strategy. The use of Vpx to destroy SAMHD1 represents a notable strategy developed by lentiviruses to thwart cellular antiviral defenses. These findings bring to light a previously unknown mechanism of innate immunity and raise the question of whether the pharmacological alteration of intracellular dNTP pools might be a therapeutic approach to treating virus infection. The implications of depleting the nucleotide pool as a host-defense mechanism are potentially far-reaching.
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