The APOBEC3G Deamination Independent Mode of HIV Inhibition
Date
2013-07-29
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Masters
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
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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.
Description
Keywords
APOBEC3G, HIV, Deamination Independent Mode
Citation
Degree
Master of Science (M.Sc.)
Department
Microbiology and Immunology
Program
Microbiology and Immunology