The identification of human apolipoprotein B mRNA-editing enzyme catalytic
polypeptide-like 3G (hA3G) represents one of the most significant advances in
retrovirus research. A3G interacts with the virion infectivity factor (Vif) of HIV-1.
This Vif-APOBEC3G axis now broads the scope of knowledge in host-virus
regulation and forms a very attractive target for development of an entirely new class
of anti-HIV drugs. Recently, a lot of progresses were made about the mechanism of
their intriguing regulation within cells, the impact of these enzymes on viral evolution
and disease progression, and their roles in controlling the replication of exogenous
retroviruses and the structure of A3G. And, there are still much room for both function
and structure research.
The first clue for this project originates from an unexpected findings of the
attempted cloning of wild-type A3G from human PBMCs. After sequencing, We
found there are 5 Non-synonymous point-mutants in the A3G coding sequence. In
order to eliminate the possibility of personal differences, We next amplified the gene
from monocytic leukemia cell line, THP-1. Surprisingly, We found that the ratio of
mutation is approximately 87%, by using the thermostable DNA polymerase from
Pyrococcus furiosus which with the error rate of 1.6x10(-6), which undoubtedly
implied that there are multiple transcripts in THP-1 cell line.
Then we tested the A3G distribution and expression level in nine cell lines by
using RT-PCR and Western blotting. Based on the results, we expressed the A3G gene
via lentivirus system in the A3G-low-expression cell lines, and silenced the A3G gene
through siRNA in the A3G-high-expression cell lines. These works have founded the
research basis for A3G-HIV-1 interactions.
Finally, we transfected the wild-type and the mutated clones of A3G (H186R、
I309T、F310S) and (W34R、Y222H、V224G、A246V、F289L) to HEK293T to find
out the impact of mutations on A3G expression level and its dynamic characters. We
concluded that the 5-point-mutant transcript can enhance the formation of High-molecular-mass A3G complexes. And both the outdated A3G could reduce the
A3G expression rate when compared with the wild-type. The significance of the
outdated transcripts and truncated form of A3G discovered in our project need further
research.
In summary, we discovered the existence of A3G multiple transcripts in THP-1.
Based on the results of RT-PCR and Western blotting, we wounded the system of
A3G expression in visit, established the essential basis for the analysis of A3G
function. We also plan to detect the functions of the other mutated forms discovered
in this project.
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