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Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels.

ABSTRACT: Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.

SUBMITTER: Amie SM

PROVIDER: S-EPMC3711331 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels.

Amie Sarah M SM Daly Michele B MB Noble Erin E Schinazi Raymond F RF Bambara Robert A RA Kim Baek B

The Journal of biological chemistry 20130605 28

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs ...[more]

PMID: 23744077

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Project description:Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Dataset Information

Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels.

Publications

Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels.

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