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Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase.

ABSTRACT: It is well established that the cytosine deaminase APOBEC3G can restrict HIV-1 virions in the absence of the virion infectivity factor (Vif) by inducing genome mutagenesis through deamination of cytosine to uracil in single-stranded HIV-1 (-)DNA. However, whether APOBEC3G is able to restrict HIV-1 using a deamination-independent mode remains an open question. In this report we use in vitro primer extension assays on primer/templates that model (-)DNA synthesis by reverse transcriptase from the primer binding site (PBS) and within the protease gene of HIV-1. We find that APOBEC3G is able to decrease the initiation of DNA synthesis by reverse transcriptase approximately 2-fold under conditions where reverse transcriptase is in excess to APOBEC3G, as found in HIV-1 virions. However, the delay in the initiation of DNA synthesis on RNA templates up to 120 nt did not decrease the total amount of primer extended after extended incubation unless the concentration of reverse transcriptase was equal to or less than that of APOBEC3G. By determining apparent Kd values of reverse transcriptase and APOBEC3G for the primer/templates and of reverse transcriptase binding to APOBEC3G we conclude that APOBEC3G is able to decrease the efficiency of reverse transcriptase-mediated DNA synthesis by binding to the RNA template, rather than by physically interacting with reverse transcriptase. All together the data support a model in which this deamination-independent mode of APOBEC3G would play a minor role in restricting HIV-1. We propose that the deamination-independent inhibition of reverse transcriptase we observed can be a mechanism used by APOBEC3G to slow down proviral DNA formation and increase the time in which single-stranded (-)DNA is available for deamination by APOBEC3G, rather than a direct mechanism used by APOBEC3G for HIV-1 restriction.

SUBMITTER: Adolph MB

PROVIDER: S-EPMC3662766 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase.

Adolph Madison B MB Webb Jonathon J Chelico Linda L

PloS one 20130523 5

It is well established that the cytosine deaminase APOBEC3G can restrict HIV-1 virions in the absence of the virion infectivity factor (Vif) by inducing genome mutagenesis through deamination of cytosine to uracil in single-stranded HIV-1 (-)DNA. However, whether APOBEC3G is able to restrict HIV-1 using a deamination-independent mode remains an open question. In this report we use in vitro primer extension assays on primer/templates that model (-)DNA synthesis by reverse transcriptase from the p ...[more]

PMID: 23717565

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Project description:BackgroundSAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro.ResultsWe first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels.ConclusionsAltogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.

Dataset Information

Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase.

Publications

Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase.

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