Retroviral Replication and Human Gene Mapping 
Patrick O. Brown, 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 ofDNA 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 J. Michael Bishop's laboratory at the Univer- 
sity 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 infection can lead to AIDS (acquired 
immune deficiency syndrome), leukemia, lym- 
phoma, or degenerative diseases of the central 
nervous system. Millions of people throughout 
the world are infected with the human immuno- 
deficiency virus, HIV, and will likely succumb to 
AIDS unless an effective treatment is developed. 
Retroviral Replication 
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 of the host cell. 
Thus the viral genome, then called a provirus, 
becomes an integral part of the cell's DNA. Inte- 
gration of a provirus into its host cell's genome is 
essential for retroviral reproduction. This dis- 
tinctive feature of the life cycle of the retrovi- 
ruses accounts for many of the characteristics as- 
sociated with retroviral infection, including 
insertional mutagenesis, induction of tumors, 
and viral latency and persistence. Moreover, the 
fact that retroviruses are designed to intro- 
duce foreign genes into cellular DNA makes 
them exceptionally useful as tools for genetic 
engineering. 
The question that propels much of the work in 
our laboratory is. How does a retrovirus get its 
DNA into a cell's nucleus and integrate it into the 
cell's DNA? To address this question, we are us- 
ing genetic, biochemical, and cytological ap- 
proaches. For example, fluorescent probes that 
bind to viral DNA enable us to track individual 
viral particles as they infect a cell. This allows us 
to investigate the influence of such factors as the 
cell division cycle and the role of specific cyto- 
skeletal elements on the entry of viral particles 
into the nucleus of cells. The methods we have 
developed to study such nuclear entry will also 
be applied in exploring the mechanisms of sub- 
cellular localization and the intracellular traf- 
ficking of intermediates in viral replication. 
Disassembly of the nuclear envelope at mitosis 
provides one possible route for nuclear entry of 
viral intermediates. Hence we are studying the 
role of mitosis in the viral life cycle. It has been 
recognized for many years that establishment of a 
retroviral provirus proceeds much more readily 
in actively dividing cells than in their resting 
counterparts, but the basis for this phenomenon 
remains unknown. To bring this phenomenon 
into clearer focus, we are investigating the de- 
pendence of specific steps in the life cycle of the 
murine leukemia virus (MLV) on the host cell's 
stage in its own division cycle. Understanding 
how cellular functions can determine the fate of 
an infecting retrovirus may lead us to new ap- 
proaches to antiviral therapy and to improve- 
ments in the use of retroviruses as vectors for 
gene therapy. 
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 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 requirements 
for the reaction. The enzymatic machinery that 
carries out integration can be isolated from in- 
fected cells in a stable complex with the uninte- 
grated viral DNA molecule. This complex was the 
focus of our initial studies of integration, and we 
are continuing to investigate its structure and 
composition. 
Our ability, however, to study the enzymology 
of integrase, the viral protein that actually cata- 
lyzes integration, was limited by the paucity of 
integrative complexes in infected cells. In the 
past year, we have constructed bacterial strains 
genetically engineered to produce abundant 
quantities of the integrase proteins from HIV and 
MLV and have developed simple purifications of 
these proteins. Using model substrates, we can 
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