VOL. 6, 1986 
RETROVIRUS PACKAGING CELL LINES 
RNA tRNA 
VIRAL CAP BINDING PACKAGING 
ENHANCERS SITE SITE SIGNAL 
ORIGIN OF 
SECOND STRAND POLY (A) 
ONA SYNTHESIS SITE 
pAM 
pPAM 
pPAM2 
pPAM3 
Sau3AI Pst I 
SA TAG 
t > 
pol, any j “ 
SA TAG 
i | 
pol, any ) r~'~; 
(Ball) 
I 
SV40 r g | T \ 
POLY (A) ' 1 ' 
SITE 
SA 
pol, any 
TAG I 
53 
BamHI (Bell) 
SA TAG 
pol, any 
(Bell) 
pPAM4 
CAP SD ATG 
SA 
i. 
gag. pol, anv 
Sau3AI SauSAI PstI 
(Bell) 
, 0.4Kb , 
FIG. 1. Packaging constructs are depicted without surrounding plasmid sequences. Large, open boxes represent retroviral LTRs; small, 
open boxes represent other retroviral sequences; and hatched boxes denote simian virus 40 sequences. Landmark restriction sites are shown, 
but all of the sites for a given enzyme are not necessarily shown. The plasmid containing a recombinant amphotropic helper virus (pAM) and 
the derivative of this virus in which the packaging signal has been removed (pPAM) have been described before (15). Important features for 
retrovirus replication are noted above the pAM construct. The construct pPAM2 was made from pPAM by replacement of the 3' LTR with 
the late polyadenylation signal from simian virus 40, isolated as a 237-base-pair BamHI to Bell fragment. The end of the retroviral genome 
was cleaved with Rsal at position 7762 (25), which cuts just upstream of the termination codon of env, and a synthetic oligonucleotide was 
added to duplicate the part of env that was removed. This oligonucleotide also contained a BamHI site downstream of the terminator codon 
for env for addition of the simian virus 40 fragment containing a polyadenylation signal. pPAM3 was made from pPAM2 by removing viral 
sequences 5' of the viral enhancers in the 5' LTR by using an SauiAl site at position -352 (25). In addition to the deletions in pPAM3, pPAM4 
has a deletion which removes the tRNA-binding site and 3' portion of the 5' LTR while preserving the splice donor site. This deletion was 
made by cleavage of the LTR at an Smal site at position 28 (25) in the R region of the LTR, addition of a BamHI linker, and linkage to an 
Sau3Al site at position 161 (25) (BamHI and SauiAl leave complementary 5’ extensions). The constructs are all carried in pBR322. pAM. 
pPAM, and pPAM2 are inserted in pBR322 in place of the Tc r gene between the Clal and BvuII sites. The Clal sites remain, but the sites at 
the other ends of the constructs were destroyed during attachment to the Pvull site of pBR322. pPAM3 and pPAM4 were inserted in place 
of the Tc r gene between BamHI and PvuII. Only the SauiM site remains at the 5' end of the construct, and the sites at the 3' end have been 
destroyed, kb, Kilobase; SD, splice donor; SA, splice acceptor. 
Inhibition of recombination to produce helper virus. We 
have previously shown that certain vectors interact with 
PA12 cells to produce helper virus (16). N2 virus (Fig. 2) and 
derivatives containing additional genes inserted at the 3’ end 
of the virus produce helper virus when introduced into PA12 
cells. Helper virus production is dependent on the presence 
of gag sequences upstream of the neo gene in this vector, as 
deletion of these sequences leads to vectors that do not 
produce helper virus (16). A plausible model for this event 
would involve copackaging of the viral vector and RNA 
derived from the pPAM packaging construct present in PA12 
cells, followed by recombination in the gag region during 
reverse transcription in an infected cell. Addition of the 5' 
portion of the vector to the packaging construct would result 
in creation of a fully replication-competent virus containing 
a vector-derived packaging signal. In contrast, two recom- 
bination events would be required to generate helper virus 
from the pPAM3 packaging construct, since defects are 
[370] 
Recombinant DNA Research, Volume 12 
