RETROVIRAL INTEGRATION 
Patrick O. Brown, M.D., Ph.D., Assistant Investigator 
Work in Dr. Brown's laboratory is focused on 
the mechanism by which a retrovirus inserts a 
DNA copy of its genome into a chromosome of 
its host cell. This integration reaction is an essen- 
tial step in retroviral replication. Retroviral inte- 
gration provides a highly efficient means of in- 
serting foreign DNA into mammalian chromosomes 
and thus has important potential for genetic engi- 
neering and gene therapy. Moreover, since in- 
tegration depends on virally encoded functions 
and has no known essential cellular counterpart, 
it is a promising target for development of novel 
antiviral agents. Two retroviruses are currently 
being studied: the Moloney murine leukemia virus 
(MLV) and the human immunodeficiency virus 
(HIV). 
I. Murine Leukemia Virus Integration and 
Nuclear Entry. 
In the past year Dr. Brown and his colleagues 
have completed a characterization of the structure 
of intermediates in the MLV integration process. By 
carrying out the integration reaction in vitro, it was 
possible to identify and recover a key intermediate 
in the joining of viral to host DNA. In this reaction 
intermediate, only one strand at each end of the 
viral DNA molecule is joined to the host DNA. Se- 
quence analysis demonstrated that this initial joint 
involves the 3' ends of the viral DNA molecule and 
the target DNA 5' ends. The detailed structure of 
this joint depends directly on the structure of the 
viral DNA precursor. It was thus possible to show 
that the linear DNA product of reverse transcription 
is joined directly to host DNA, without first being 
circularized, as had previously been believed. The 
unintegrated linear viral DNA molecule was also 
characterized. The ends of this molecule are ini- 
tially blunt but are soon processed by removal of 
two bases from each 3' end. This processing event 
is necessary to expose the specific 3 -OH group that 
is used in joining the viral DNA to its target. A virus 
carrying a defective allele of the int gene, and thus 
unable to carry out integration, was shown to be 
incapable of processing the viral DNA 3' ends. This 
result suggests that cleavage of the terminal two 
bases from the 3' ends of the linear precursor is 
one essential role that the Int protein plays in inte- 
gration. Since this cleavage event is temporally and 
spatially separated from the joining of viral to target 
DNA, it is not likely to provide the energy for forma- 
tion of the new bonds linking viral and target DNA, 
yet earlier studies by Dr. Brown and his colleagues 
had shown that no external source of energy is re- 
quired for integration. It follows that the formation 
of bonds joining viral and target DNA is probably 
energetically coupled to cleavage of the target DNA 
molecule. This hypothesis and the chemical mecha- 
nism of the joining reaction are currently under in- 
vestigation. 
Characterization of the native state of uninte- 
grated viral DNA in acutely infected cells has dem- 
onstrated that the DNA is in a large (160 S) nucleo- 
protein complex that includes all the activities 
required for efficient integration. This complex, 
with its integration activity intact, can be efficiently 
immunoprecipitated using antisera specific for the 
viral capsid protein. Moreover, while the viral DNA 
in the complex can be cut with restriction endonu- 
cleases, the resulting fragments remain associated 
with a 160 S particle. These data suggest that this 
intracellular form of the virus has significant simi- 
larities to the core of the extracellular virus particle. 
Further studies directed at the identification of the 
components of this complex and definition of its ar- 
chitecture are under way. It is likely that this nu- 
cleoprotein complex has important roles in addi- 
tion to its role in integration. For example, it may 
play an active part in directing the transport of the 
viral genome into the nucleus of the infected cell. 
Studies that focus on the mechanism by which the 
viral nucleoprotein complex gains access to the nu- 
cleus are currently in progress in Dr. Brown's labo- 
ratory. 
II. HIV Integration. 
HIV is unique among the retroviruses both in its 
importance as a cause of human disease and in the 
complexity of its life cycle. Dr. Brown and his col- 
leagues have developed methods for studying the 
integration of HIV in a cell-free system and have 
begun the biochemical characterization of this pro- 
cess. Preliminary experiments have established the 
conditions necessary for activity and shown that the 
in vitro reaction can be carried out in the absence 
of an extrinsic energy source and that the enzy- 
matic machinery required for HIV integration is in a 
nucleoprotein complex with the viral DNA. Further 
characterization of the molecular mechanism of 
Continued 
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