Retroviral Life Cycle

Fundamental to the utility of retroviral vectors are the particular characteristics of the retroviral life cycle, illustrated in Fig. 1. The initial steps of the life cycle, from binding of the virus to a target cell through integration of its genetic material into that cell's genome, do not require the de novo synthesis of any viral proteins. Accordingly, a retroviral particle can be used as a vector to deliver genetic material without the requirement for any viral protein synthesis or infectious particle production.

The retroviral genome codes for 3 basic polyproteins (Fig. 2A), produced by alternate splicing of the RNA transcript, which are further processed into their component proteins. The gag gene products produce the protein core of the viral particle, which encapsidates 2 copies of the linear RNA genome. Also associated with the viral core are the products of the pol gene, the enzymes involved in particle maturation (protease) and DNA metabolism (reverse transcriptase and integrase). The env gene product is a glycoprotein that is anchored in the plasma membrane of the host cell. It is therefore incorporated into the lipid envelope that surrounds the retroviral particle as a result of its budding from a host cell. The budding process does not kill the host cell, which allows for the establishment of stable producer cell lines that continuously release retroviral vector particles.

Table 1 Gene Therapy Applications

1. Gene replacement/augmentation

Especially suited to single gene defects and discrete populations of target cells (e.g., ADA deficiency, cystic fibrosis)

2. Suicide/toxic genes

To eliminate certain cells (e.g., HSV thymidine kinase for cancer cells)

3. Protective genes

Expression of the gene product renders the cell resistant to viral attack (e.g., intracellular antibodies, antisense constructs for HIV gene therapy)

4. Immune stimulation

To stimulate the host's immune system, in particular, to cancer cells (e.g., HLA genes, immune stipulatory cytokines)

5. Cell marking

For autologous bone marrow transplantation in cancer therapy (e.g., neomycin)

A retrovirus binds to a new host cell by virtue of the interaction of the Env glycoprotein with an appropriate cellular receptor. This interaction triggers a series of events that ultimately lead to the fusion of the lipid envelope surrounding the virus with the target cell membrane. Entry of the retroviral core into the cell allows the reverse transcriptase enzyme to copy the viral RNA genome into a double-stranded DNA provirus, which is then randomly inserted into a host chromosome through the action of the integrase protein. Certain sequences in the RNA genome are essential for packaging, reverse transcription, and integration to occur, and are highlighted in Fig. 2B.

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