2 research outputs found
Computational Model and Dynamics of Monomeric Full-Length APOBEC3G
APOBEC3G (A3G) is
a restriction factor that provides innate immunity
against HIV-1 in the absence of viral infectivity factor (Vif) protein.
However, structural information about A3G, which can aid in unraveling
the mechanisms that govern its interactions and define its antiviral
activity, remains unknown. Here, we built a computer model of a full-length
A3G using docking approaches and molecular dynamics simulations, based
on the available X-ray and NMR structural data for the two protein
domains. The model revealed a large-scale dynamics of the A3G monomer,
as the two A3G domains can assume compact forms or extended dumbbell
type forms with domains visibly separated from each other. To validate
the A3G model, we performed time-lapse high-speed atomic force microscopy
(HS-AFM) experiments enabling us to get images of a fully hydrated
A3G and to directly visualize its dynamics. HS-AFM confirmed that
A3G exists in two forms, a globular form (ā¼84% of the time)
and a dumbbell form (ā¼16% of the time), and can dynamically
switch from one form to the other. The obtained HS-AFM results are
in line with the computer modeling, which demonstrates a similar distribution
between two forms. Furthermore, our simulations capture the complete
process of A3G switching from the DNA-bound state to the closed state.
The revealed dynamic nature of monomeric A3G could aid in target recognition
including scanning for cytosine locations along the DNA strand and
in interactions with viral RNA during packaging into HIV-1 particles
Nanoscale Characterization of Interaction of APOBEC3G with RNA
The
human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor
of the HIV-1 virus in the absence of viral infectivity factor (Vif).
The molecular mechanism of A3G antiviral activity is primarily attributed
to deamination of single-stranded DNA (ssDNA); however, the nondeamination
mechanism also contributes to HIV-1 restriction. The interaction of
A3G with ssDNA and RNA is required for its antiviral activity. Here
we used atomic force microscopy to directly visualize A3GāRNA
and A3GāssDNA complexes and compare them to each other. Our
results showed that A3G in A3GāRNA complexes exists primarily
in monomericādimeric states, similar to its stoichiometry in
complexes with ssDNA. New A3GāRNA complexes in which A3G binds
to two RNA molecules were identified. These data suggest the existence
of two separate RNA binding sites on A3G. Such complexes were not
observed with ssDNA substrates. Time-lapse high-speed atomic force
microscopy was applied to characterize the dynamics of the complexes.
The data revealed that the two RNA binding sites have different affinities
for A3G. On the basis of the obtained results, a model for the interaction
of A3G with RNA is proposed