Cel. Vol. 33. 1 S3- 159. May 1963. Copyright C 1963 by MfT 
0O92-6674/63/O1S3C7 S02.00/0 
Construction of a Retrovirus Packaging 
Mutant and Its Use to Produce 
Helper-Free Defective Retrovirus 
Richard Mann* , l Richard C. Mulligan', and 
David Baltimore*' 
* Whitehead Institute for Biomedical Research 
Cambridge, Massachusetts 02139 
’ Center for Cancer Research 
and Department of Biology 
Massachusetts Institute of Technology 
Cambridge, Massachusetts 02139 
Summary 
A mutant of Moloney murine leukemia virus (M- 
MuLV), pMOV-f , was constructed by deletion of 
about 350 nucleotides from an infectious proviral 
DNA clone between the putative env mRNA 5' splice 
site and the AUG that initiates the coding sequence 
for PrfiS** 9 . Although the parent wild-type proviral 
clone, pMOV-i/'*, quickly causes a high level of re- 
verse-transcriptase-containing virus particles to be 
released from transfected NIH/3T3 cells, transfec- 
tion of pMOV-V-" into these cells initially results in 
very little release. By 9 to 10 days after transfection, 
however, pMOV-f"-transfected cells produce infec- 
tious virus. Thus pMOV-^~ has a defect that can be 
repaired in transfected NIH/3T3 cells, presumably 
by recombination with a sequence normally present 
in the cells. Cell lines with pMOV-^~ stably inte- 
grated into chromosomal DNA produce reverse-tran- 
scriptase-containing particles that lack detectable 
M-MuLV RNA but the cells efficiently complement 
replication-defective, packagable retroviruses. Thus 
pMOV-f~ has a defect in the packaging of genomic 
RNA into virions but can provide in trans the prod- 
ucts necessary for virion production. The deletion In 
pMOV-V' - appears to define a site required in c/s for 
packaging of MuLV RNA into virions. Cell lines car- 
rying pMOV-f can be used to produce helper-free 
stocks of natural or synthetic defective retroviruses. 
Introduction 
Retrovirus RNA is packaged into virions as a 70S dimer of 
two identical, capped and polyadenylated 35S RNAs (for 
review see Varmus, 1 982). How the virus achieves selec- 
tive packaging of these RNAs and excludes other mRNAs 
remains one of the more obscure aspects of the retrovirus 
life cycle. One possible explanation is that there is a site 
on the viral genome which interacts with a virion protein to 
direct specifically the packaging of the RNA. If such a site 
were deleted from a viral genome, the result would be a 
c/s defect preventing the encapsidation of genomic RNA. 
Such a mutant, however, should still be capable of direct- 
ing the synthesis of all viral proteins. 
In searching for a c/s packaging signal we noted that 
retroviruses package full-length RNA but not the spliced 
env mRNA, even though these two RNAs have identical 
5' and 3' termini (Gerwin and Levin. 1977; Rothenberg et 
al., 1978; Levin and Setdman, 1979; Stoltzfus and Kuhnert, 
1979). The ability of the virus to differentiate between these 
RNAs suggested that an essential part of a packaging 
signal might be in the region spliced out to form the env 
mRNA. If such a site was outside the coding region for 
PtSS 9 * 0 and reverse transcriptase, it would have to be 3' 
of the presumed splice donor site and 5' of the AUG that 
initiates translation of Pr65 1 ’* t '. 
A second due to the location of a packaging site came 
from the study of a Rous sarcoma virus-transformed quail 
cell line that is deficient in packaging viral RNA (LiniaJ et 
al., 1978). This mutant has a 150 bp deletion somewhere 
between 300 and 600 bp from the left end of the provirus 
thereby implicating once again the region upstream from 
the start of PT65 9 * 0 as important for packaging (Shank and 
Linial. 1980). A complicating property of this mutant is that 
it is /rans-dominant: wild-type genomes are also ineffi- 
'ciently packaged upon superinfection, suggesting that a 
second mutation exists in some "packaging factor" which 
relaxes the normal specificity for Rous sarcoma virus RNA 
(LiniaJ et al., 1978; Linial, 1981). 
In this study, we describe the deletion from Moloney 
murine leukemia virus (M-MuLV) of approximately 350 bp 
between the left long terminal repeat (LTR) and the start 
codon for PT65 1 ** 9 . The resulting mutant has a ds-active 
deficiency for packaging of genomic RNA and can be 
used to generate pure stocks of defective retroviruses, 
including recombinant retroviral vectors. Watanabe and 
Temin (1982) have provided evidence for a similar site in 
an avian retrovirus. 
Results 
Construction of pMOV-^“ 
To examine the function of sequences upstream from the 
start of Pr65 !> * 0 and downstream from the env mRNA splice 
donor site in M-MuLV. we deleted these sequences from 
a doned DNA representation of the genome. As detailed 
in Figure 1 , the deletion was made from a Bal I site to a 
Pst I site, and a Hind 111 site was generated at the point of 
deletion. Based on the published sequence of M-MuLV 
(Shinnick et al., 1981), the size of the deletion was 351 
bp. The 5' deletion endpoint was only 6 bp from the 
presumed donor site for the env mRNA splice, and the 3' 
deletion endpoint was approximately 50 bp from the start 
codon for Pr65 p *° (Figure 2). The resulting deleted plasmid 
is termed pMOV-^ - . 
Virus Spread Is Defective after Transfection of 
pMOV-yT 
To determine whether pMOV-f generated transmissible 
virus, it and its wild-type counterpart, pMOV-^"*, were 
independently transfected into NIH/3T3 cells, and reverse 
transcriptase in the culture supernatants was assayed over 
time. By 4 days after transfection, the medium from a 
culture transfected with pMOV-^" had 3-fold more reverse 
transcriptase activity than that of a standard MuLV pro- 
ducer line, MOV-1 (Figure 3). We interpret this rapid irv 
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Recombinant DNA Research, Volume 12 
