penetrates a cell, the viraJ RNA is first 
converted to DNA, the DNA enters the 
nucleus and integrates randomly into a 
chromosome, becoming indistinguish- 
able from any other cellular gene (see 
Fig. 1). It is from this integrated form, 
the provirus, that the viral genes are ex- 
pressed. Progeny virus are formed 
which leave the cell by budding from the 
cell membrane. It is important to point 
out that the integration of the viral 
genome into the cell’s chromosome is 
an essential pan of its replication. With 
a few exceptions, the presence of the 
viral genome in the infected cell, the ex- 
pression of its genes, and the formation 
of progeny virus have no apparent ef- 
fect on the viability of the infected cell. 
Thus, retroviruses have features that 
make them particularly suitable for 
gene transfer. By replacing the viraJ 
genes with the gene of interest and using 
the efficient viral integration process, it 
is now possible to transfer a gene into 
the infected cell as if it were a viral gene. 
How does this work? The viral gen- 
ome, encoded in an RNA molecule in 
the virion and in a double stranded 
DNA in the infected cell, contains two 
types of information which can be class- 
ified as cis and trans functions. The 
trans functions are the viral proteins 
such as the polymerase and the envelope 
glycoprotein. The cis functions are the 
various signals scattered throughout the 
viral genome, such as the promoter and 
enhancer sequences required for initia- 
tion of RNA transcription. Other ex- 
amples of cis functions are the various 
sequences which direct the integration 
of the viral genome into the chro- 
mosome of the infected cell as well as 
the encapsulation signal (VO required for 
virus packaging. To construct a virus 
that can be used as a vector, the cis func- 
tions must be retained while the trans 
functions can be replaced by the gene of 
interest. What is thereby created is a 
replication defective retrovirus missing 
its own genes. The trans functions 
which were removed can be supplied by 
another virus (called a helper virus) in 
the same cell or by a second defective 
virus that still contains the trans func- 
tions missing in the vector. This 
mechanism for obtaining viability by 
combining the activities of two defective 
components (commonly used in 
Vol. 4. No 6(1986) 
Fijure 1. The retroviral lifr cycle. After adsorption and uptake (i.e.. binding and infection) of the 
viral genome, the stngle-sirandedlSS) RNA is convened to double-stranded (DS) DNA by reverse 
transenptase. After uptake into the nucleus, the DNA integrates randomly into a chromosome to 
form the provirus. The provirus serves as a template both for mRNAs to yield viral proteins and 
for new. full length genomic RNA. The genomic RNA and viral proteins combine and. by bud- 
ding. release a new infectious virus to repeat the cycle (Reprinted with permission from A. Bernstein 
tt at. in Genetic Engineering: Principles and Methods, p. 235. Plenum. NY, 1985 .) 
somatic cell hybridization) is called 
complementation. Thus, the retroviral 
vector carrying the gene of interest can 
be assembled into a virion, exit from the 
cell, infect a target cdl, and, through the 
cis functions retained in the vector, the 
foreign gene is transferred into a 
chromosome of the cell as if it were a 
viral gene. 
In the laboratory, this process takes 
place in two steps as shown in Fig. 2. 
First, a preparation of retrovirus is 
generated (by, for example, transfection 
of a packaging cell line, see below) that 
contains the foreign gene (this is, 
therefore, a recombinant virus): second, 
the gene is delivered to the target cell by 
infection of the target cells with the 
recombinant virus. Recombinant DNA 
techniques allow the manipulation of 
DNA sequences, but not RNA sequen- 
ces. Therefore, the initial procedure is 
to combine the portions of the retroviral 
DNA carrying the cis functions, with 
the DNA fragment carrying the foreign 
gene (and removing in the process the 
viral trans functions). How is this vec- 
tor DNA converted into a correspond- 
ing vector RNA and encapsidated into 
a virus? To do this, the vector DNA is 
introduced by the standard (however in- 
efficient) DNA transfection procedures 
into a specially designed cell line called 
a packaging cell (18. 22). The packag- 
ing cell line harbors a virus that is defec- 
tive in the cis function that is required 
to be present for the viral RNA to be 
able to encapsidate into a virion. All its 
trans functions are normal, however, so 
that they will complement those func- 
tions missing from the incoming vector 
DNA. It is then possible to identify and 
isolate the rare cells that have taken up 
the vector DNA by use of a selectable 
gene present in the vector. For example, 
cells infected with a vector containing a 
Neo* gene are resistant to toxic levels 
of the neomycin-like antibiotic, G418. 
The vector DNA not only is tran- 
scribed into RNA which is translated 
within the cell, but also it is transcribed 
into viral genomic RNA which is encap- 
sidated into a retroviral virion, and 
secreted into the medium. The recom- 
binant virus carrying the foreign gene 
can now infect a target cell and then in- 
tegrate into its genome, carrying the 
foreign gene with it. Since the viral 
RNA carrying the trans functions can- 
not encapsidate, the virions carry only 
vector RNA. These particles can infect 
only once since the viral RNA they con- 
tain have no trans function genes. The 
particles are a one-time-only delivery 
system. 
At present, it is now possible to insert 
a gene into a retroviral vector, obtain 
recombinant virus, and then infect 
target cells and express the foreign gene 
from the cells’ chromosomes. What has 
proven more difficult is to make this 
process efficient. An efficient gene 
transfer system is desirable for its use- 
fulness to basic research, but is an abso- 
lute prerequisite for application to 
human therapy (1). Indeed, a large ef- 
BioTechniques SOS 
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Recombinant DNA Research, Volume 12 
