Retroviral Replication 
Patrick O. Broum, M.D., Ph.D. — Assistant Investigator 
Dr. Brown is also Assistant Professor of Pediatrics and of Biochemistry at Stanford University School of 
Medicine. He received his B.A. degree in chemistry from the University of Chicago. His graduate work with 
Nicholas Cozzarelli at the University of Chicago was focused on the mechanisms of DNA topoisomerases. 
He received his Ph.D. and M.D. degrees from the University of Chicago, completed a pediatrics residency 
at Children's Memorial Hospital in Chicago, and then joined Michael Bishop's laboratory at the University 
of California, San Francisco. There he began to investigate the mechanism of retroviral integration, 
which has continued to be the major focus of his research. 
RETROVIRUSES are an important cause of dis- 
ease in most vertebrate species. In humans, 
retroviral infections can lead to AIDS (acquired 
immune deficiency syndrome), leukemia, lym- 
phoma, and degenerative diseases of the central 
nervous system. Millions of people are infected 
with the human immunodeficiency virus, HIV, 
and will likely succumb to AIDS unless an effec- 
tive treatment is developed. 
The retroviral genes are carried in the virus 
particle as RNA molecules. When the virus infects 
a cell, it transcribes these molecules, its RNA ge- 
nome, into a double-stranded DNA molecule and 
inserts this into a chromosome in the nucleus of 
the host cell. Thus the viral genome, then called a 
provirus, becomes an integral part of the cell's 
genome. Integration of a provirus into its host 
cell's DNA is essential for retroviral reproduc- 
tion. This distinctive feature of the retroviral life 
cycle accounts for many of the characteristics as- 
sociated with retroviral infection, including in- 
sertional mutagenesis, induction of tumors, and 
the latent and persistent nature of many retroviral 
infections. Moreover, the fact that retroviruses 
are designed to introduce foreign genes into cel- 
lular DNA makes them exceptionally useful as 
tools for genetic engineering. 
How does a retrovirus get its DNA into a cell's 
nucleus and integrate it into the cell's DNA, and 
how does the cell regulate these processes? 
To investigate the molecular mechanism by 
which a retrovirus inserts its DNA into that of the 
infected cell, we have developed a variety of 
methods for studying the retroviral integration re- 
action in a test tube. We have used this approach 
to define several discrete steps in the joining of 
viral to cellular DNA and to determine the re- 
quirements for the reaction. The enzymatic ma- 
chinery that carries out integration can be iso- 
lated from infected cells in a stable complex with 
the unintegrated viral DNA molecule. We are 
currently using electron microscopy as well as 
biochemical methods to define the structure of 
this viral replication intermediate. 
To study the enzymology of integrase, the viral 
protein that actually catalyzes integration, we 
have constructed genetically engineered bacte- 
rial strains that produce abundant quantities of 
the integrase proteins from HIV and murine leu- 
kemia virus and have developed simple purifica- 
tions of these proteins. Using small synthetic DNA 
molecules as model substrates, we can now 
readily study their catalytic activities, which in- 
clude the sequence-specific processing of the 
ends of the viral DNA and the joining of these 
ends to a target DNA molecule. We have made 
progress in the past year toward understanding 
the organization of integrase and how it binds the 
viral and target DNA substrates. For example, we 
have identified DNA substrates that function pref- 
erentially either as donors or as targets in a DNA 
joining assay. By studying the competition of 
these DNAs for binding to the enzyme, we have 
identified two distinct sites that bind viral and 
target DNA, respectively. By analyzing the rever- 
sal of the usual DNA joining reaction, we have 
discovered that integrase has a previously unrec- 
ognized DNA splicing activity. We are investigat- 
ing the possibility that this new activity may play 
a role in integration and in the high-frequency 
recombination that occurs between viral ge- 
nomes during replication. 
The structure of integrase is clearly of central 
importance to understanding integration. Be- 
cause efforts to obtain conventional crystals of 
integrase have been unrewarding, we are prepar- 
ing to use electron diffraction methods to deter- 
mine a structure from two-dimensional crystals. 
By constructing specific mutants and defining 
their biochemical defects, we have begun to 
identify the functions of specific protein regions. 
To extend this approach, we are developing a 
new genetic system that we hope will enable us 
to screen for functional defects among millions of 
mutant integrases, based on their ability to carry 
out recombination in bacteria. This system 
should also facilitate our efforts to develop genet- 
ically altered integrases with properties more fa- 
vorable for therapeutic applications. For exam- 
ple, we would like to develop an integrase that 
53 
