The APOBEC3G Deamination Independent Mode of HIV Inhibition

Abstract

ABSTRACT APOBEC3G (Apo3G) is a host cell restriction factor of viruses that produce a singlestranded (ss) DNA replication intermediate (Sheehy et al., 2002; Suspene et al., 2004). Apo3G is studied primarily for its ability to restrict propagation of the retrovirus, HIV. In cell culture, Apo3G can only inhibit HIV if it lacks its virion infectivity factor (Vif). The host-pathogen interface between Apo3G and HIV has become a new target of study for the development of novel HIV therapeutics (Prochnow et al., 2009; Sheehy et al., 2003). Apo3G induces mutagenesis of the HIV proviral DNA (Mangeat et al., 2003; Zhang et al., 2003). Apo3G has the ability to induce transition mutations, i.e. cytosine to thymine, through deamination of cytosine to form uracil. Deamination activity induces numerous mutations that causes gene inactivation of the HIV provirus thus restricting the HIV lifecycle. Apo3G attenuates HIV virion infectivity in the absence of the virion infectivity factor (Vif) by inducing genome mutations through deamination of cytosine to uracil in HIV minus strand DNA. Independent from deaminase activity, Apo3G may also interfere with HIV reverse transcription by preventing full length cDNA from forming (Iwatani et al., 2007), nucleocapsid (NC) mediated strand annealing (Guo et al., 2007; Guo et al., 2009; Li et al., 2007), and RNaseH activity of the reverse transcriptase (Li et al., 2007). Whether Apo3G is able to restrict HIV by a deamination-independent mode remains controversial. In particular, the existence of the deamination independent mode was challenged since the Apo3G deamination null mutant E259Q was shown to have limited or no ability to inhibit HIV-1 replication (Schumacher et al., 2008). This research assesses the ability of Apo3G to inhibit reverse transcription of HIV genomic RNA. It is hypothesised that based on the ability of Apo3G to bind and oligomerize on single stranded nucleic acids (Chelico et al., 2008), and its high affinity for RNA (Chelico et al., 2010), that Apo3G can inhibit RT mediated primer extension as well as nucleocapsid mediated strand annealing. Additionally, it is hypothesized that Apo3G cannot inhibit RT RNAseH activity, as Apo3G has been shown to have a low affinity for DNA/RNA hybrids (Iwatani et al., 2006). We will test these hypotheses by using in vitro assays that mimic in vivo reverse transcription events. Here we have shown that Apo3G is able to decrease the efficiency with which HIV-1 reverse transcriptase synthesizes DNA from an RNA primer annealed to an RNA template. Apo3G had a minimal affect on primer initiation and primarily inhibits primer elongation. Using iii the monomeric mutant, F126A/W127A, we show that the deamination independent mode of inhibiting reverse transcriptase is impaired without oligomerization on template RNA. We also provide evidence that the Apo3G mutant E259Q should not be considered a deamination null proxy for native Apo3G since it exhibits decrease in RNA binding affinity compared to the native form. We did not find that Apo3G inhibited HIV NC-mediated strand annealing activity or RNaseH activity of HIV-1 reverse transcriptase. The data suggest a two-tiered mechanism for inhibition of reverse transcriptase-mediated DNA synthesis that is dependent upon 1) the ability of Apo3G to oligomerize on RNA substrates and 2) bind RNA with high affinity. Ascribing a mechanism to the deamination independent mode of HIV-1 restriction by Apo3G suggests that the enzyme may use this mechanism in vivo to delay completion of proviral DNA synthesis which, may negatively impact the HIV-1 lifecycle.