The Retroviral Life Cycle

1. The Retroviral Particle

The retroviral genome within the virus particle is composed of two identical RNA molecules. Retroviral vectors are often derived from the murine virus, Moloney leukemiavirus (MoMLV), the genome ofwhich contains three open reading frames called gag pal, and env. These reading frames are bound by regions that contain the signals essential for expression of the viral
genes. At the 5’ end of the RNA genome is the R and U5 region, followed by a short stretch of noncoding DNA before the start of the gag region. Gag encodes the structural proteins of the viral capsid, the polregion encodes the enzymatic activities for genome processing, and the env region specifies the proteins of the viral envelope. To the 3’ side of the coding sequences lie additional regulatory sequences, the U3 region, and a copy of the R region. A single copy of the RNA genome, as found in the virion particle, is shown in Fig. 1. Retrovirus Vectors

2. Infection of the Target Cell
The virus particle contains two RNA strands forming a complex with viral enzymes (polproducts) and host tRNA molecules within a core structure of gug proteins. Surrounding this capsid is a lipid bilayer derived from the host-cell membrane, containing the viral envproteins. These envproteins bind to the cellular receptor for the virus, and the particle enters the host cell,
probably via a process of receptor-mediated endocytosis.

3. Reverse lknscription of the Viral RNA into DNA
Following shedding of the outer envelope, the viral RNA is copied into DNA by the process of reverse transcription. This takes place within the RNA/protein complex and is catalyzed by the reverse transcriptase enzyme of the pal region.
Reverse transcriptase uses as a primer a molecule of hostcell tRNA, which is carried into the infected cell in the particle and is homologous to a region just downstream of the 5’ U5 region (-PBS). It commences its activity by copying the donor viral genome into DNA, moving into the U5 and R regions (toward the left end of the genome) (Fig. ZA). Since the copying extends to
the extreme 5’ end of the RNA template, a second enzymatic activity of reverse transcriptase digests away the portion of the RNA molecule that has already been copied (U5 region). This liberates the DNA copy (of U5-R) to base pair, via the R sequence, to another genomic RNA molecule, the acceptor (6), (Fig. ZB). F o 11o wing this rntermolecular “jump” of template by reverse transcriptase, the growing DNA molecule (minus strand) is extended by copying the acceptor RNA genome. It moves out of R, through the U3 region, and beyond, past a second Primer Binding Site (tPBS) that lies at the boundary of U3. The minus strand is elongated through the env, pal, and gag regions as far as the tRNA primer binding site of the acceptor RNA genome. Meanwhile, a second molecule of reverse transcriptase becomes primed, and it commences DNA synthesis of a plus strand of DNA at the tPBS site on the minus strand (Fig. ZC). The plus strand is elongated to copy the U3-RUS-(- PBS) cassette from the minus strand. Now, an zntramoleculur templatejump occurs, by which the plus-strand USR-U5-(-PBS) cassette is transposed to the 3’ end of the minus strand in such a way that the -PBS on the plus
strand is matched with the -PBS of the minus strand (Fig. 2D) . The generation of the linear duplex DNA molecule is now relatively straightforward. The minus strand is completed by extension into the U5-R-U3 region of the plus strand; the plus strand is completed by copying the gug$ol-enuW-R-U5 region of the minus strand. The final products of these complex molecular maneuvers are linear duplex DNA molecules with identical boundary regions