Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Diamond, Tracy L.; Roshal, Mikhail; Jamburuthugoda, Varuni K.; Reynolds, Holly M.; Merriam, Aaron R.; Lee, Kwi Y.; Balakrishnan, Mini; Bambara, Robert A.; Planelles, Vicente; Dewhurst, Stephen; Kim, Baek

doi:10.1074/jbc.m408573200

Cited by 270 publications

(456 citation statements)

References 38 publications

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“…We quantified deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP) concentration using the published protocols. 19,20 The concentration of all the tested dNTPs was reduced by about seven-fold in quiescent HepG2 cells but remained high in HepG2.2.215 cells (Fig. 4A).…”

Section: Resultsmentioning

confidence: 97%

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Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2009

Hepatology

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Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 10 7 to 10 11 virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA-transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene. Conclusion: Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV-host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs. (HEPATOLOGY 2010;51:1538-1546 H epatitis B virus (HBV) is a widespread pathogen responsible for acute and chronic hepatitis and is a causative factor of hepatocellular carcinoma (HCC). 1 HBV is a small-enveloped noncytopathic virus containing a circular partially doublestranded DNA genome, and exhibits strong tropism for human liver cells. 2 During replication, the viral polymerase reverse-transcribes the pregenomic RNA to DNA using deoxyribonucleotide triphosphates (dNTPs). HBV preferentially replicates in nondividing cells, 3 in which the concentration of dNTP is low, which raised the question whether dNTP concentration is adequate to support viral yield. The level of dNTPs in a nondividing adult liver cell is <0.4 lM. 4 The Michaelis constant (K m ) of the viral polymerase at a dNTP concentration of 0.4 lM is only 62%, whereas a 4 lM concentration gives 93.3% activity. 5 Furthermore, one hepatocyte produces 50-300 hepatitis B virions per day, 6 and because the HBV genome is approximately 3.2 kilobases, between 3 Â 10 5 to 2 Â 10 6 dNTPs are consumed per day in this process. Considering cell volume as 500 fL, 7 a resting cell contains approximately 1.2 Â 10 5 dNTP molecules. Thus, the total amount of dNTPs used for HBV production per day exceeds the amount found in a nondividing hepatocyte. Because HBV does not activate the cell cycl...

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Section: Resultsmentioning

confidence: 97%

“…15 Enzymatic Assay for dNTPs Measurement. dNTP extraction is based on 19 and dNTP level was quantified by DNA polymerase fill-in reaction as described. 20 …”

Section: Methodsmentioning

confidence: 99%

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2009

Hepatology

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“…These cells differ in the concentrations of their intracellular deoxyribonucleoside triphosphates (dNTPs). Activated CD4 ϩ T cells, which are dividing cells, contain high levels of dNTPs (1-16 M) as compared with terminally differentiated macrophages (20 -40 nM) (3,4). Recently, we and others reported that the low dNTP pool in macrophages is partially due to active hydrolysis of cellular dNTPs by SAMHD1, a cellular dNTP triphosphohydrolase (5,6).…”

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confidence: 99%

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

Daddacha

Noble²,

Nguyen

et al. 2013

Journal of Biological Chemistry

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Background: Under limiting dNTP concentrations, HIV-1 RT incorporates rNTPs during DNA synthesis. Results: HIV-1 RT utilizes dNTP less efficiently around rNMPs, and mismatch extension fidelity is significantly reduced. Conclusion: Presence of an rNMP in DNA template slows HIV-1 RT-mediated DNA synthesis and reduces fidelity. Significance: This study provides insight into how rNMP incorporation during proviral DNA synthesis can affect HIV-1 replication kinetics and fidelity.

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“…In particular, HIV-1 reverse transcription is much slower in monocyte-derived macrophages (MDMs) than in activated T cells. This difference is attributed mainly to the limited availability of nucleotide precursors in these nondividing cells, in which the average deoxynucleoside triphosphate (dNTP) concentration (∼26 nM) is about 200 times lower than in PHA-activated T cells (10)(11)(12). The dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) decreases dNTP levels by hydrolyzing dNTP into deoxynucleosides (dNs) and triphosphates, thereby limiting HIV-1 reverse transcription in dendritic cells, macrophages, and resting T cells (13)(14)(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway

Allouch

David

Amie

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Macrophages are a major target cell for HIV-1, and their infection contributes to HIV pathogenesis. We have previously shown that the cyclin-dependent kinase inhibitor p21 inhibits the replication of HIV-1 and other primate lentiviruses in human monocyte-derived macrophages by impairing reverse transcription of the viral genome. In the attempt to understand the p21-mediated restriction mechanisms, we found that p21 impairs HIV-1 and simian immunodeficiency virus (SIV)mac reverse transcription in macrophages by reducing the intracellular deoxyribonucleotide (dNTP) pool to levels below those required for viral cDNA synthesis by a SAM domain and HD domain-containing protein 1 (SAMHD1)-independent pathway. We found that p21 blocks dNTP biosynthesis by down-regulating the expression of the RNR2 subunit of ribonucleotide reductase, an enzyme essential for the reduction of ribonucleotides to dNTP. p21 inhibits RNR2 transcription by repressing E2F1 transcription factor, its transcriptional activator. Our findings unravel a cellular pathway that restricts HIV-1 and other primate lentiviruses by affecting dNTP synthesis, thereby pointing to new potential cellular targets for anti-HIV therapeutic strategies.

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Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Cited by 270 publications

References 38 publications

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway

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