( ameplasmid ensures that expression ...)Safe Production of High-Titer Retrovirus

Replication-Defective Retroviral Gene Transfer Systems

Fig.1

Nonreplicating retroviral vectors contain all of the cis elements required for transcription of mRNA molecules encoding a gene of interest and packaging of these transcripts into infectious virus particles (Figure 1). The vectors typically comprise an E. coli plasmid backbone with the gene of interest inserted into a pair of 600-bp viral long terminal repeats (LTRs). The LTR is divided into three regions. The U3 region, containing the retroviral promoter/enhancer, is flanked in the 3 direction by the R region, which contains the viral poly-adenylation signal (pA). The U5 region follows and, along with R, contains sequences that are critical for reverse transcription. Expression of the viral RNA is initiated within the U3 region of the 5 LTR and is terminated in the R region of the 3 LTR. Between the 5 LTR and the coding sequence for the gene of interest resides an extended version of the viral packaging signal (Y+), which is required in cis for the viral RNA to be packaged into virion particles.

To generate infectious virus particles that carry the gene of interest, specialized packaging cell lines have in the past been generated that contain chromosomally integrated expression cassettes for viral gag, pol, and env proteins, all of which are required in trans to make virus. The gag gene encodes internal structural proteins, pol encodes reverse transcriptase (RT) and integrase, and the env gene encodes the viral envelope protein. The envelope protein resides on the viral surface and faci
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