Yasumasa Iwatani & Judith
G. Levin talk with ScienceWatch.com and answer a
few questions about this month's Fast Breaking Paper in the
field of Biology & Biochemistry. The authors have
also sent along a PowerPoint presentation of their
Article Title: Deaminase-independent inhibition of
HIV-1 reverse transcription by APOBEC3G
Y;Chan, DSB;Wang, F;Maynard, KS;Sugiura,
W;Gronenborn, AM;Rouzina, I;Williams, MC;Musier-Forsyth,
Journal: NUCL ACID RES
Year: DEC 2007
* NICHD, Mol Genet Lab, NIH, Bethesda, MD 20892 USA.
* NICHD, Mol Genet Lab, NIH, Bethesda, MD 20892 USA.
(addresses have been truncated)
Why do you think your paper is highly
The human protein APOBEC3G (A3G), a cytidine deaminase, was identified as a
host defense factor that blocks HIV-1 reverse transcription and virus
replication in the absence of the viral protein Vif. A3G is currently under
active investigation by a large group of researchers because it has
potential application in AIDS therapy. For this reason, work in the field
is focused on elucidating the mechanism of A3G's antiviral activity and
papers that contribute new insights into this process receive a lot of
Judith G. Levin
Most of the studies reported in the literature have been performed
primarily with cell-based systems and/or with unfractionated enzyme derived
from viral lysates. Since it is difficult to dissect individual events that
occur during the course of virus infection in cells, we chose a more
biochemical approach to clarify the effect of A3G on reverse transcription
at the molecular level.
To perform these experiments, we took advantage of defined biochemical
assay systems that we developed over the years for studies on viral DNA
synthesis and used purified proteins: A3G (see below); HIV-1 reverse
transcriptase (RT); and HIV-1 nucleocapsid protein (NC), which we and
others have shown to increase the efficiency and specificity of reverse
Our A3G paper was the first to show that A3G inhibits all RT-catalyzed DNA
elongation reactions, but not RNase H activity or NC's ability to promote
annealing. These results could be explained by critical differences in the
nucleic acid binding properties of A3G, RT, and NC, as measured by
single-molecule DNA stretching and fluorescence anisotropy. Figure 1 shows
how A3G binding to single-stranded nucleic acid acts as a roadblock to
RT-catalyzed DNA polymerization during reverse transcription.
Does it describe a new discovery, methodology, or
synthesis of knowledge?
This work describes a new discovery. Since A3G's deaminase activity was not
required for inhibition of polymerization reactions in our system, our
findings provide a novel mechanism for deaminase-independent A3G-mediated
antiviral activity that has also been observed in infected cells. This type
of mechanism might also explain how A3G and other members of the human
APOBEC3 family inhibit replication of retrotransposons, Hepatitis B virus,
and Adeno-associated virus, without a requirement for deamination.
Would you summarize the significance of your paper in
All animals, including humans, have cellular defense factors, which fight
viral and bacterial infections that are harmful to the host. Often these
defense factors are not enough and drugs (or vaccination) must be employed
to obtain a favorable outcome. This is also the case for HIV-1 infection.
In 2002, scientists in the United Kingdom identified a human protein,
called APOBEC3G (A3G), which can block HIV replication under certain
conditions. Since A3G is a potential inhibitor that could be used in AIDS
therapy, this protein is the subject of intense investigation in the field.
A3G is a "cytidine deaminase," which means it is an enzyme that converts
cytidine residues (one of the four bases in DNA) into uridines (not a
normal constituent of DNA).
There is strong evidence indicating that deaminase activity is crucial for
A3G's antiviral activity. However, a portion of the antiviral activity can
also be deaminase-independent. We investigated the mechanism for A3G's
inhibitory effect using purified proteins and a cell-free system to study
the relationship between A3G and viral DNA synthesis. The data showed that
inhibition was deaminase-independent in this case.
Based on the binding properties of the key proteins in our system, we
concluded that when A3G binds to single-stranded DNA or RNA templates, it
acts as a roadblock to hinder polymerization reactions that occur during
reverse transcription (see Figure 1). This is the first study to propose a
mechanism for the deaminase-independent antiviral activity of A3G.
How did you become involved in this research, and were
there any problems along the way?
Our group has been engaged in research on reverse transcription for many
years. We initiated the A3G project because of our strong interest in host
factors that influence this process. Moreover, we were intrigued by early
work showing that in the absence of the viral protein Vif, a cellular
factor inhibits HIV-1 reverse transcription. This factor later turned out
to be human A3G.
We decided to take advantage of the reconstituted systems that we had
developed for our studies on HIV-1 reverse transcription to determine the
mechanism of A3G inhibition. To do this, we had to first produce large
amounts of catalytically active, highly purified A3G. Our decision to
express and purify A3G was an enormous challenge, since we knew that many
people had tried to do this and failed. We also endured fruitless attempts
to obtain functional protein from E. coli.
The key to success turned out to be the use of a baculovirus expression
system and the total commitment and determination of Dr. Yasumasa Iwatani,
then a postdoctoral fellow in the laboratory, who performed this work. The
molecular characterization of the human A3G protein is described in Iwatani
et al., "Biochemical Activities of Highly Purified, Catalytically
Active Human APOBEC3G: Correlation with Antiviral Effect," J.
Virol. 80:5992-6002, 2006.
Where do you see your research leading in the
In our work on A3G thus far, we have demonstrated that the availability of
a pure protein (uncontaminated by either host or viral proteins) has made
it possible to perform a rigorous analysis of A3G's molecular properties,
which complements in vivo assays that measure antiviral activity.
The success of this approach has encouraged us to pursue additional
biochemical studies of A3G, in conjunction with cell-based mutational
analysis and structure-function studies.
Do you foresee any social or political implications for
The global AIDS pandemic continues to endanger the health of people
worldwide and it is now estimated that 40 million people are currently
infected with HIV. Research on A3G is critical for developing new antiviral
strategies to combat this devastating disease. To achieve this goal, it is
urgent that we obtain a more detailed understanding of A3G's antiviral
activity and identify new targets for drug discovery and design. It is also
vital to have sufficient funding to ensure the success of these efforts.
NOTE: This figure is available in an
PowerPoint file. When viewing, use "Slide Show"
Dr. Yasumasa Iwatani
Chief, Laboratory of Infectious Diseases
Clinical Research Center
National Hospital Organization Nagoya Medical Center
Nagoya, Aichi, Japan
Dr. Judith G. Levin
Chief, Section on Viral Gene Regulation
Laboratory of Molecular Genetics
Eunice Kennedy Shriver National Institute of
Child Health and Human Development
National Institutes of Health
Bethesda, MD, USA
Keywords: human protein APOBEC3G, cytidine deaminase, host
defense factor, blocks HIV-1 reverse transcription, viral protein Vif,
AIDS therapy, HIV-1 reverse transcriptase, HIV-1 nucleocapsid protein,
RT-catalyzed DNA elongation reactions, single-molecule DNA stretching,
fluorescence anisotropy, deaminase-independent antiviral activity of