The Principles of Retroviral Vector Construction

To achieve the aims of catiuge and expression of nonviral genes, the construction strategy must encompass two considerations:

1. The vector must be able to behave as aviral genome to allow it to pass as a virus from the producer cell line. Hence, its DNA must contain the regions of the wild-type retroviral genome required in cis for incorporation in a retroviral particle.
2. The vector must contain regulatory signals that lead to the optimal expression of the cloned gene once the vector is integrated in the target cell as a provirus. These may or may not be provided by viral DNA sequences.

1. Manipulation of the Vector as DNA

Vectors are constructed as part of bacterial plasmid DNA molecules (e.g., pBR322), which can be grown in bacteria in the usual way. As such, they can be manipulated using standard techniques for recombinant DNA
The vectorcontaining plasmids were synthesized initially from a proviral clone of MoMLV, by using, or creating, suitable restriction sites to remove or insert relevant DNA sequences. The DNA sequences surrounding the virusspecific DNA contain as few restriction sites as possible, to simplify the insertion of foreign sequences into unique restriction sites in the constructs. The following discussions will focus only on the viral sequences within the plasmid.

2. Propagation of the Vector as a Virus

The finding that retroviruses can transduce cellular genes suggested that the LTRS do not discriminate which genes they express. In fact, it has been shown that all the viral genes can be discarded and replaced by exe
genous coding sequences. However, consideration of the life cycle highlights those signals that must be incorporated into the vector DNA These essential sequence elements are:

1. The Y sequence that ensures the packaging of the vector DNA into virions.This sequence corresponds to nucleotides 215365 in the MoMLV sequence (10). More recent vector constructs contain the extended Y sequence, which extends up to nucleotide 1039, incorporating the start of the grzg gene (Yt), but with the AUG start codon of the viral gene mutated,
2. The tRNA binding site that is necessary to prime reverse transcription of the RNA form of the vector (in the virion) into DNA within the target cell (-PBS) ;
3. The sequences in the LTRs that permit the “jumping” of the reverse transcriptase between RNA strands during DNA synthesis;
4. Specific sequences near the ends of the LTRs that are necessary for the integration of the vector DNAinto the hostcell chromosome in the ordered and reproducible manner characteristic of retroviruses (13);
5. The sequences adjoining the 3’ LTR that serve as the priming site for synthesis of the plus-strand DNA molecule (tPBS) .