Mutant 
LTR 
B 
M-MuLV 
MSVgpt 
MSV 
xtoi p f ,i 
_i_i_ 
Xhol 
W 
Xbol Xbol 
Xbol 
Xbol 
Xbbt 
6gl n Hind ID Xhol- 
-4 US*. 
fOl 
.xboitr-^ 
got 
Pul 
j J 
Xhol 
i 
W 
Xbol^-Hpol xt .°i 
0.5 Kb 
Fijut 4. Structure ol pMSVgpt and Its Use to 
Assay the Helper Activity ol Rettovral Constructs 
(A) Recombinant plasmid pMSVgpt is shown. Se- 
quences derived from retrovruses are ndealed 
by ttvcfr bnes. with the long termmal repeats (LTRs) 
as fried bores. The gpt gene (Muligan and Berg. 
1980) e nckcated by an open bor Mr* ceAiar 
DMA Ranking the recombnant retrovrus is (id- 
eated by a wavy line. The polyoma virus early 
regen (Magnusson and IMUson. 1977) (base pars 
2932 to 4632) is indicated as a dashed line, and 
p6R322 sequences derived from the "person mi- 
nus" derivative (Lusky and Botchan, 1981) are 
indicated by a thn bne. 
(B) Structure and ongri of the provirus segment of 
pMSVgpt showng that no intact retrovfral genes 
are present. The reason for nduding the Xba I to 
Xba I fragment of M-MuLV in pMSVgpt is irrelevant 
to the present use of the plasmid. The MSV restric- 
tion map s from Vande Woude et al. (1979). 
(C) Protocol for assessing the helper activity of 
retrovral constructs. 
c 
transfect 
IO M g HELPER PLASMIO 
I Ml pMSVgpt 
INFECT WITH SUPERNATANT 
24 Mrs POST -TRANSFECTION 
TWO 
DAYS 
SELECT FOR 
gpt' 18-10 Hoys) 
pMOV-vT or pMOV-*‘ into NIH/3T3 cells (Figure 4C). After 
24 hr. culture supernatants of the transfected cells were 
used to infect a second plate of NIH/3T3 cells. Two days 
following the infection, selection pressure for XGPRT was 
applied, and 8-10 days later gpt* colonies (colonies con- 
taining the gpt gene) were counted. pMOV-^* yielded 2.5 
x 1 0' gpt* colony forming units (cf u) per milliliter of culture 
supernatant, and pMOV-^ - yielded 5 x 10 3 gpt* efu per 
milliliter (Table 1). No gpt* efu were detectable when 
pMSVgpt was transfected by itself. Thus pMOV-^ - could 
efficiently provide all functions in trans required for viable 
virus production. 
Construction of Cell Lines With pMOV-^~ Stably 
Integrated and Expressed 
To explore further the properties of pMOV-^ - , we decided 
to establish cell lines containing the 4>~ mutant stably 
integrated into the chromosome. These lines were made 
by the cotransfection of pMOV-^ - and pSV2gpt, a SV40 
hybrid vector capable of XGPRT expression (Mulligan and 
Berg, 1980). Cells from gpt* colonies obtained in this way 
were cloned and established into 3 lines, f-1, ^-2 and f- 
3. All three lines had detectable reverse transcriptase 
activity in the culture supernatant (Table 2), indicating that 
they all contained sufficient MuLV information to express 
and bud reverse-transcriptase-containing particles. Only 
the supernatant from ^-3 was infectious: infections with 1 
[354] 
Table 1. Assay ol helper activity ol pMOVV* and pMOV-^ - 
Helper FRasmid 
gpr (clu/ml)* 
pMOV-f* 
2.5 x 10* 
pMOV-*- 
5 x 10* 
none 
<10 
* gpT colony (orrang inrts per muter ol transfected cel culture superna- 
tant 24 hr alter transfection. 
ml of culture supernatant from either ^-1 or \F2 onto NIH/ 
3T3 cells yielded no reverse transcriptase producer cells 
even after 3 weeks of culture. Thus the ^-3 cells were 
producing a nondefectrve virus, wherease the ^-1 and 
2 cells were not. 
Viral RNA In ^-2 Cells Is Poorly Packaged Into 
Virions 
Because the \f>~ mutant appeared defective in a function 
required only in c/s, we suspected RNA of missing 
sequences important for its packaging into virus partides. 
To address this question directly, total cellular RNA was 
purified from the MOV-1 (an M-MuLV producer) and \p-2 
cell lines, and viral RNA was extracted from purified parti- 
cles released from the MOV-1 and f-2 cells. The MOV-1 
and i/-2 viral RNAs were derived from virion preparations 
adjusted to contain equal amounts of reverse transcriptase 
Recombinant DNA Research, Volume 12 
