Virion-associated Uracil DNA Glycosylase-2 and Apurinic/Apyrimidinic Endonuclease Are Involved in the Degradation of APOBEC3G-edited Nascent HIV-1 DNA

Abstract

Cellular cytidine deaminases APOBEC3 family is a group of potent inhibitors for many exogenous and endogenous retroviruses. It has been demonstrated that they induce G to A hypermutations in the nascent retroviral DNA, resulting from the cytosine (C) to uracil (U) conversions in minus-stranded viral DNA. In this report, we have demonstrated that the result of C to U conversion in minus-stranded DNA of human immunodeficiency virus type 1 (HIV-1) could trigger a degradation of nascent viral DNA mediated by uracil DNA glycosylases-2 (UNG2) and apurinic/apyrimidinic endonuclease (APE). Since antiviral activity of APOBEC3G is partially affected by UNG2 inhibitor Ugi or UNG2-specific short-interfering RNA in virus-producing cells but not target cells, the virion-associated UNG2 most likely mediates this process. Interestingly, as APE-specific short-interfering RNA can also partially inhibit the anti-HIV-1 activity of APOBEC3G in virus-producing cells but not in target cells and APE molecules can be detected within HIV-1 virions, it seems that the required APE is also virion-associated. Furthermore, the in vitro cleavage experiment using uracil-containing single-stranded DNA as a template has demonstrated that the uracil-excising catalytic activity of virion-associated UNG2 can remove dU from the uracil-containing viral DNA and leave an abasic site, which could be further cleaved by virion-associated APE. Based upon our observations, we propose that the degradation of APOBEC3G-edited viral DNA mediated by virion-associated UNG2 and APE during or after reverse transcription could be partially responsible for the potent anti-HIV-1 effect by APOBEC3G in the absence of vif.APOBEC3 is a family of cytidine deaminases that has been identified as the host factor to restrict various retroviruses, endogenous retroviruses, and long interspersed nucleotide element (LINE) elements (1-11). It is closely related to APOBEC1, a cytidine deaminase that causes a specific cytosine to uracil change in the apolipoprotein B mRNA, and to an activationinduced deaminase (AID) enzyme that causes hypermutation of immunoglobulin genes (12). The similarities of the catalytic domains in these proteins strongly suggest that APOBEC3 edits the nucleic acids of various retrotransposons. Among these cytidine deaminases, APOBEC3G is the most extensively studied enzyme. It can be efficiently incorporated into human immunodeficiency virus type 1 (HIV-1) 4 particles and causes extensive cytosine to uracil conversion in the viral minus-stranded DNA during reverse transcription (2, 3, 13). The significant C to U conversion in minus-stranded DNA is apparently correlated with the decreased viral infectivity. Two consequences could occur after the C to U conversion. First, uracil in minusstranded DNA could be excised by recruited uracil DNA glycosylase-2 (UNG2), a host DNA repair enzyme. The resulting abasic site could be further cleaved by apurinic/apyrimidinic endonucleases (APE). The vif-defective viruses generated from the restrictive cells are unable to effe...

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

“…One well-characterized mechanism is the enzymatic deamination of cytosine in the minus strand of HIV-1 cDNA by members of the APOBEC3 subfamily (APOBEC3D, -F, -G, and -H) to generate C/G→U/A transition mutations after plus strand synthesis (43,44). This deamination mechanism leads to lethal hypermutation of the viral genome and potent restriction of viral infection, but there are conflicting reports as to whether UNG2 excision of uracils is part of the restriction mechanism (45)(46)(47). In contrast, the dUTP-mediated mechanism elaborated here results in nonmutagenic incorporation of uracil on both strands of the viral DNA and requires hUNG2 to prevent viral integration.…”

Derepression of MicroRNA-mediated Protein Translation Inhibition by Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G (APOBEC3G) and Its Family Members

“…1a). These plasmids were transfected into 293T cells, which naturally do not express A3G (22,27), with or without an A3G-HA-expressing plasmid. Fig.…”

“…A3G can be incorporated into HIV-1 particles and cause extensive C to U conversion in the viral minusstranded DNA during reverse transcription (24 -26), which can trigger its degradation by virion-associated uracil DNA glycosylase-2 (UNG2) and apurinic/apyrimidinic endonucleases (APE) or lethal hypermutation in the HIV-1 genome (26,27). However, accumulating evidence indicates that A3G protein carrying mutations in the catalytic domain of the cytidine deaminase retains substantial anti-HIV-1 activity (24, 28 -31).…”

Human APOBEC3G Can Restrict Retroviral Infection in Avian Cells and Acts Independently of both UNG and SMUG1

“…However, it is notable that although some reports have indicated a role for the UNG as well as possibly the SMUG1 uracil DNA glycosylase (42,50), APOBEC3-mediated restriction is actually unaffected by a deficiency of UNG activity (24,34). Consistent with this result, we find that expression of Ugi, a potent and irreversible inhibitor of UNG, in human 293T cells does not influence the antiviral properties of APOBEC3G on HIV ⌬Vif infection whether the inhibitor is expressed in the producer cells and/or in the target cells (Fig.…”