Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs

Abstract

By using a retroviral construct derived from Moloney murine leukemia virus and capable of expressing the dominant selectable neo gene, we measured the effects of moving or deleting a sequence (psi) known to be required in cis for the packaging of genomic RNA into virus particles. When psi was at its wild-type position (in SVX virus) near the 5' end of the RNA, the titer of infectious virus production was 5 X 10(6) G-418-resistant CFU per ml. The titer was decreased approximately fivefold when psi was moved, in its proper orientation, to near the 3' end of the virus (SVX-psi C) and was decreased approximately 600-fold when psi was moved, in its proper orientation, into the U3 region of the long terminal repeat. When psi was deleted (SVX-psi-) or inserted in the opposite orientation at either of these two positions, the titer was decreased by 3000-fold relative to SVX. In SVX-psi C, psi was no longer in the intron (as it is in SVX and Moloney murine leukemia virus) but was moved to a region which is only exonic. This resulted in the encapsidation of both spliced and unspliced RNAs, their efficient reverse transcription, and their integration into the genome of an infected cell. A number of proviruses resulting from integration of either spliced or unspliced RNAs were cloned. Four of these clones were subjected to sequence analysis in the region of the splice sites, and it was determined which sites are used by these viruses and also which are used by Moloney murine leukemia virus.