HIV-1 is one of the most well known viruses in the world. The difficulty in combating infection is the rather prevalent mutagenic nature of the virus, which in turn means that anti-retrovirals are quickly rendered inactive after these mutations occur. In addition to this mutagenic nature, HIV-1 has a wide array of accessory proteins that further aids in successful infection. In a recent study by the Journal of Biological
Chemistry, researchers focused on one of these accessory HIV-1 proteins, the curious viral protein, Viral Infectivity
Factor (Vif). Vif itself is a rather small 23- kilodalton protein that plays a
pivotal role in HIV replication. Vif’s main function is to counteract the antiviral
activity of APOBEC3G by tagging it via an E3 ubiquitin ligase and targeting it
for proteasomal degradation (Wang 2013). APOBEC3G belongs to a superfamily of
APOBEC proteins, which are known to play a large role in innate anti-viral
immunity (Harris 2003). APOBEC3G is a certain protein of this family that is
responsible for causing hypermutations in the progeny viral DNA. It is a single
stranded deoxycytidine deaminase, that, when Vif is not present, incorporates
itself into the budding virion through binding of the (+) viral RNA strand. APOBEC3G functions by catalyzing the
deamination of cytosine to uracil, thus a deaminase enzyme. Subsequently, this causes reverse
transcriptase to transcribe the newly converted uracil as adenine in the viral
RNA transcript. This mutation is sufficiently able to mutate the pro viral DNA,
and inactivate it (Romani 2009).
Interaction of APOBEC3G/Vif |
The
paper focuses on the processivity of APOBEC3G, and how it is affected when in
the presence of Vif. Vif was primarily seen to inhibit APOBEC3G by targeting it through proteasomal degradation, as well as down-regulating APOBEC3G mRNA translation, so the ability for it to inhibit its function by another mechanism provides another pathway for anti-retroviral therapy. Past research has shown that APOBEC3G scans its substrate by both sliding and microscopic
jumping around the ssDNA, providing a thorough 3D scan that increases its
efficiency in locating its deamination target (Chelico 2006). In this paper, the researchers investigate the novel molecular mechanism
of degradation-independent Vif mediated inhibition of APOBEC3G. Two Vif
variants were used; VifIIIB and VifHXB2, both are seen to
inhibit APOBEC3G deaminase activity, and in this experiment both provide a
novel way in which Vif inhibits deaminase activity by successfully altering the
scanning mechanism employed by APOBEC3G.
In
the first experiment, the researchers constructed a model HIV-1 replication
system to measure any change in APOBEC3G mutagenesis in the presence of the Vif
variants. First they performed an assay with only APOBEC3G and saw that in
comparison with just reverse transcriptase, APOBEC3G caused a 17-fold increase
in population mutation frequency, and a 11-fold increase in the clone mutation
frequency. Having a baseline of the amount of mutations that APOBEC3G induces,
the researchers then performed two additional assays, this time one of each Vif
variant was introduced alongside APOBEC3G. From here they plotted the
percentage of clones having a mutation in comparison with their location on the
transcript. The histogram presented really makes it easy to
understand the data. The histogram, Figure 1, shows that without Vif, APOBEC3G usually has
a high range of induced mutations, 30% of recovered clones had 12 mutations or
greater per transcript, as well as about 25% of clones showing 6-8 mutations
per transcript. In the presence of VifHXB2, 55% of clones showed 3-5
mutations per transcript, while there was no more clones with 12 mutations or
greater. The VifIIIB variant also showed reduction of clone
mutations, to a lesser effect, by a shift down to 3-8 mutations per transcript.
Together the data shows that both Vif variants are able to inhibit APOBEC3G
induced mutagenesis, and their potential for being targeted for therapy.
Figure 1. Population Distribution of Mutations |
The previous results showed that although APOBEC3G deamination was being inhibited by the Vif variants, each affected inhibition differently. To investigate the specificity of the particular Vif variants, the researchers looked at the interaction between the scanning mechanism of APOBEC3G and the Vif variants. To measure this the researchers conducted an assay measuring APOBEC3G catalyzed deaminations along a synthetic DNA substrate. The assay utilizes a 118-nucleotide long DNA substrate, which contains two CCC deamination motifs 61 nucleotides apart. The researchers assessed the processivity of the scanning by using a processivity factor number. The processivity factor of APOBEC3G, without the presence of Vif, is 9.1, which means that APOBEC3G is nine times more likely to undergo a deamination at both CCC deamination motifs along the same DNA substrate, rather than deaminating at both sites in separate single stranded DNA. The assay was performed again in the presence of the Vif variants, the results showed that the processivity of APOBEC3G was reduced to 4.1 in the presence of VHXB2 and 5.6 in the presence of VIIIB.
As aforementioned, APOBEC3G behaves in a
highly processive way in that it is able to scan and jump around the
transcripts to find the deamination motifs. The researchers decided to run an
assay that could aid in discerning how the different Vif variants cause this
decrease in APOBEC3G processivity. The assay consisted of adding a
complementary DNA oligonucleotide in between the two CCC motifs. This new
partially double stranded DNA segment is unable to bind with APOBEC3G, and thus
the sliding characteristic in scanning is inhibited. However, since APOBEC3G
can disassociate and reassociate, microscopic jumping, it can transverse the
blocking. The results showed that this was the case, although the processivity
factor of APOBEC3G decreased with the double stranded DNA segment, it was still
able to deaminate both 5’ and 3’ motifs due to this jumping. When VifHBX2
was added, the processivity factor dropped from 4.5 to 1.1 and almost no double
deamination events were seen. Thus the researchers speculated that VifHBX2
is responsible for inhibiting the jumping component of APOBEC3G. The researchers then performed a similar assay, but in
this instance they used an RNA/DNA hybrid section between motifs, which would
be the sort of transcript that is seen during HIV-1 replication. The assay
produced similar results as the double stranded DNA segment assay, verifying
that APOBEC3G retains its ability to slide, but cannot jump in the presence VifHBX2.
VifIIIB was seen to decrease the sliding mechanism, and thus
APOBEC3G was less capable of inducing mutations in motifs close to each other.
The results from these experiments allow
the researchers to confidently say that they have found a novel mechanism for a
secondary effect that Vif has in the presence of APOBEC3G besides just targeting it for proteasomal degradation. Through
studying the inhibition of the deaminase activity of APOBEC3G, the researchers
found that a protein-protein interaction between APOBEC3G and Vif is
responsible in altering the processivity of APOBEC3G scanning, leading
to inhibition. It is through this function that the researchers have come up with some
ideas for therapeutic use. This could be another pathway that scientists could exploit, possibly coming up with Vif inhibitors as part of a drug cocktail.
Primary Source:
Yuqing,
Feng and Robin P. Love. “HIV-1 Viral Infectivity Factor (Vif) Alters Processive
Single-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G.” The
Journal of Biological Chemistry 288 (2013): 6083-6094.
Additional References:
Chelico, L., Pham, P., Calabrese, P., and Goodman, M. F.
“APOBEC3G DNA Deaminase Acts Processively on Single-stranded DNA.” Nat. Struct.
Mol. Biol. (2006).13: 392–399.
Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK,
Watt IN, Neuberger MS, Malim MH: "DNA Deamination Mediates Innate Immunity to
Retroviral Infection". Cell (2003). 113:803–809.
Wang,
Xiaodan et al. “Interactions Between HIV-1 Vif and Human ElonginB-ElonginC are Important
for CBF-β Binding to Vif.”
Journal of Retrovirology 10 (2013).
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