Wednesday, December 18, 2013

New Mechanism Found That Adds Another Vector For Anti-Retroviral Therapy!

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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