Vector comprising a replication competent HIV-1 provirus and a heterologous gene

A vector comprising an HIV segment and a heterologous gene segment, which produces a replication competent and an infective HIV virus is disclosed. When the heterologous gene is a marker gene, the spread of the virus can be observed in both in vitro and in vivo systems. The use of this vector in establishing methods for screening anti-viral compounds is also disclosed.

The present invention is directed to a vector comprising a replication 
competent HIV-I provirus and a heterologous gene, and the use of this 
vector. Most preferably, the heterologous gene is a marker gene that can 
be used to trace HIV infection. 
The human immunodeficiency virus (HIV-I, also referred to as HTLV-III, LAV 
or HTLV-III LAV) is the etiological agent of the acquired immune 
deficiency syndrome (AIDS) and related disorders [Barre-Sinoussi, et al., 
Science 220:868-871 (1983); Gallo et al, Science 224:500-503 (1984); Levy 
et al., Science 225:840-842 (1984); Popovic et al., Science 224:497-500 
(1984); Sarngadharan et al., Science 224:506-508 (1984); Siegal et al., N. 
Engl. J. Med. 305:1439-1444 (1981)]. The disease is characterized by a 
long asymptomatic period followed by progressive degeneration of the 
immune system and the central nervous system. Studies of the virus 
indicate that replication is highly regulated, and both latent and lytic 
infection of the CD4 positive helper subset of T-lymphocytes occur in 
tissue culture [Zagury et al., Science 231:850-853 (1986)]. The expression 
of the virus in infected patients also appears to be regulated as the 
titer of infectious virus remains low throughout the course of the 
disease. Molecular studies of the regulation and genomic organization of 
HIV-I show that it encodes at least 7 genes [Ratner et al., Nature 
313:277-284 (1985); Sanchez-Pescador et al., Science 227:484-492 (1985); 
Muesing et al., Nature 313:450-457 (1985); Wain-Hobson et al., Cell 
40:9-17 (1985)]. Three of the genes, the gag, pol and env genes are common 
to all retroviruses. However, the genome also encodes six additional genes 
that are not common to most retroviruses, the vif, tat, rev (also referred 
to as art and trs), 3' nef, vpr and vpu genes [Sodroski et al., Science 
231:1549-1553 (1986); Arya et al., Science 229:69-73 (1985); Sodroski et 
al., Science 227:171-173 (1985); Sodroski et al., Nature 321:412-417 
(1986); Feinberg et al., Cell 46:807-817 (1986) Wong-Staal et al, AIDS 
Res. and Human Retroviruses 3: 33-39 (1987); and U.S. patent application 
Ser. No. 193,321 filed May 12, 1988, which are all incorporated herein by 
reference]. 
Most of these genes encode products that are necessary for the viral life 
cycle. 
The tat gene encodes a 14 kD protein that is critical for HIV replication 
and gene expression [U.S. patent application Ser. No. 806,263, filed Dec. 
6, 1985; Rosen, C. A., et al., Nature 319:555-559 (1986); Sodroski, J. et 
al., Science 227:171-173 (1985); Arya et al, Science 229: supra, Sodroski, 
et al., Science 229, supra and Dayton, A., et al., Cell 44:941-497 (1986) 
which are all incorporated herein by reference]. However, mutations 
eliminating the ability of this gene to express a functional product can 
be complemented in trans in cell lines that constitutively express the tat 
protein. 
Another gene necessary for replication is the rev gene. [U.S. patent 
application Ser. No. 865,151, filed May 20, 1986; Sodroski, et al., Nature 
321:412-417 (1986), which are both incorporated herein by reference]. 
However, this gene although necessary for production of structural 
proteins such as the envelope protein is not necessary to produce 
functional regulatory proteins such as the tat gene product and the 3' nef 
gene product, which can be made by rev-defective proviruses. 
The vif gene although not absolutely necessary encodes a 23 kD protein 
important for virus infectivity [Fisher, A. G., et al, Science 237: 
888-893 (1987); Strebel, K., et al, Nature 328:728-730 (1987)]. 
The 3' nef gene, which is located at the 3' end of the viral genome 
immediately following the env gene and overlapping the 3' LTR, encodes a 
27 kD protein which has not yet been determined to be necessary for either 
the infectivity or cytopathicity of the virus [Terwilliger, E., et al, J. 
Virol. 60:754-760 (1986); Luciw, P. A., et al, PNAS 84:1434-1438 (1987)]. 
Although a great deal of research has been expended on understanding this 
retrovirus, there have been problems with fully understanding its life 
cycle. As aforesaid, individuals infected with the virus typically exhibit 
a lengthy asymptomatic period during which viral titers are low and levels 
of T4+ lymphocytes are normal. This phase usually extends over a period of 
years. Further, the virus does not cause infection in most animals. 
Although chimpanzees can be infected, it is very difficult to follow the 
virus in chimpanzees, as they do not readily display signs of infection 
and very little viral protein or RNA is made. 
Accordingly, it would be extremely useful to have a vector containing a 
replication competition HIV provirus with a heterologous gene whereby it 
would be possible to track the route of infection in animals and in 
infected tissue at a time when very little viral protein or RNA is made. 
Further, it would be very useful to use such a vector in a system where the 
virus can be followed in vitro to screen for drugs that inhibit infection. 
SUMMARY OF INVENTION 
We have now discovered a vector comprising a sufficient number of 
nucleotides corresponding to an HIV genome to express HIV gene products 
necessary for viral replication and infectivity (HIV segment) and also 
containing a sequence of nucleotides corresponding to a heterologous gene 
(heterologous gene segment). This vector is sometimes referred to herein 
as a replication competent provirus. This vector can be used to trace the 
activities and functions of the virus in both in vivo and in vitro 
systems. 
Preferably, the vector comprises an HIV segment which does not correspond 
to a group of non-essential nucleotide sequences from the HIV virus. 
Preferably, the non-essential sequences that the HIV segment does not 
correspond to are in the 3' end of the HIV genome, and more preferably, 
the HIV segment does not contain nucleotide sequences corresponding to the 
3' nef sequences. Typically, the nucleotide sequences corresponding to the 
heterologous gene are inserted in the HIV segment where those 
non-essential sequences would appear. Preferably, the heterologous gene 
segment corresponds to a marker gene such as the chloramphenicol 
acetyltransferase (CAT) gene or a growth hormone such as human growth 
hormone (hGH). 
Preferably, the vector is not more than one kilobase larger than the HIV 
genome, more preferably the size is no more than 900 bases larger, still 
more preferably it is no more than 750 bases larger, and most preferably 
it is no more than 700 bases larger.

DETAILED DESCRIPTION OF THE INVENTION 
We have now produced a vector containing a sufficient number of nucleotides 
corresponding to an HIV genome to express HIV gene products necessary for 
viral replication and infectivity (referred to as the HIV segment) and a 
sequence of nucleotides corresponding to a heterologous gene (referred to 
as the heterologous gene segment). This vector is sometimes referred to as 
a replication competent provirus or provirus. 
Preferably, the HIV segment corresponds to nucleotides of the HIV-1 or 
HIV-2 genomes. More preferably, the HIV segment corresponds to nucleotides 
of the HIV-1 genome. However, it preferably does not correspond to many 
nucleotide sequences that are not necessary for viral replication and 
infectivity. This is because it is preferable that the vector is no larger 
than one kilobase greater than the HIV genome which it corresponds to. 
More preferably, the size of the vector is no more than 900 nucleotide 
bases larger than the HIV genome. Still more preferably, the vector is no 
more than 750 nucleotide bases larger than the HIV genome. Even more 
preferably, the vector is no more than 700 nucleotide basese larger than 
the HIV genome. Accordingly, the non-essential nucleotides that the 
vector's HIV segment should correspond to can vary depending upon the size 
of the heterologous gene segment. Preferably, the heterologous gene 
segment should be about 1 kilobase or less, more preferably it should be 
less than about 800 nucleotides. 
We have found that by using this vector the heterologous gene will follow 
the course of infection and replicaiton by the virus and can therefore be 
used as a means of identifying viral infectivity and viral function. 
The HIV segment does not correspond to a small group of non-essential 
nucleotide sequences from the HIV genome. Preferably, the non-essential 
sequences that the HIV segment does not correspond to are non-essential 
sequences in the 3' end of the HIV genome. More preferably, the HIV 
segment does not correspond to the 3' nef sequences. For example, the HIV 
segment does not have to correspond to the sequences in, for example, 
HIV-1 between the unique Xba I site and a Kpn I site located 60 
nucleotides 5' to the beginning of the 3' LTR (See FIG. 1). However, there 
are other nucleotide sequences that the HIV segment can also not 
correspond to. 
Any heterologous gene can be used so long as it will not disrupt the 
replication of the virus. More preferably, the segment corresponds to a 
gene whose expression can readily be traced so that it is possible to 
follow the replication of the virus. This would include marker genes such 
as the CAT gene, genes for growth hormones such as the human growth 
hormone gene, and many other genes well known to the person of ordinary 
skill in the art. 
For example, by using a vector comprising an HIV segment and a CAT segment 
it is possible to transfect an infectious HIV virus expressing a 
functional CAT gene. Since CAT activity is an extremely sensitive enzyme 
marker this permits studies of infection by the virus that have not been 
possible. This result occurs because in a cell transfected with this 
vector, e.g. a Jurkat cell, CAT activity can be detected using as little 
as 10 .mu.l of the infected culture. Accordingly, it is possible to 
indirectly detect viral infection at early stages where the synthesis of 
viral products is occurring at extremely low levels that are difficult to 
directly measure by looking for the presence of the marker. Use of such a 
vector permits rapid analysis of tissues that are infected by the viral 
segment as well as measurement of the level of virus expression in 
different organs. This vector is also useful in studies of superinfection, 
as well as following the course of virus infection in vivo and in vitro. 
The heterologous segment can be inserted anywhere in the vector so long as 
it does not disrupt a reading frame which expresses a necessary viral 
function. Preferably, the heterologous gene segment is inserted in the 
vector in the portion of the HIV segment wherein the non-essential 
sequences would have appeared, for example, the 3' nef sequence. However, 
this is not necessary and the heterologous gene segment can be inserted in 
numerous other reading frames in the vector besides where the 3' nef 
sequence would appear. For example, it is possible to cause a deletion in 
the 3' nef sequence to reduce the size of the HIV segment and insert the 
gene elsewhere in the HIV segment. The only caution that must be taken in 
preparing the vector is that sequences necessary for viral replication are 
not disrupted by the deletion or the insertion. However, this can readily 
be determined by empirical tests. 
In preparing a vector according to the present invention many techniques 
are possible. For example, one can use a proviral genome for the HIV 
segment and foreign DNA segments for the heterologous gene segment or one 
can chemically synthesize the HIV and heterologous gene segments. Other 
techniques known to the skilled artisan based upon the present disclosure 
can also be used to prepare the vector. 
In one method one can use a plasmid containing an infectious HIV proviral 
clone, delete a non-essential sequence, for example, the 3' nef sequence 
and insert a DNA segment for a heterologous gene in the deleted region to 
produce a vector according to the present invention. For example, an 
infectious proviral clone derived from pHXBc2 [Fisher, A. G., et al., 
Nature 316:262-265 (1985)] which contains an artificially introduced 
restriction enzyme cleavage site near the 3' end of the gene encoding the 
envelope transmembrane protein [Lobel, L. I., PNAS 81:4149-4153 (1984)] 
and referred to as pHXBc2-Xba I can be used, although any replication 
competent HIV proviral plasmid can be used therefore. This mutation 
introduced a unique Xba I site, as well as a termination signal at a 
position 17 codons before the natural termination signal of the envelope 
glycoprotein (see FIG. 1). The presence of this linker insertion did not 
alter the kinetics of virus replication as compared to the pHXBc2 parent. 
Sequences in this provirus between the unique XbaI site and a Kpn I site 
located 60 nucleotides 5' to the beginning of the 3' LTR were removed, 
resulting in a mutant provirus (pHXB-.DELTA.3'), which contains a deletion 
of 280 nucleotides including the 3' end of the env gene, the 3' nef 
initiation codon and 220 nucleotides of the 3' nef gene. Mutant proviruses 
equivalent to this one can readily be constructed by the person of 
ordinary skill in the art by using standard techniques. Typically, one 
uses known restriction enzyme sites to delete and/or insert the various 
sequences. Although the 3' nef coding sequences extend beyond the Kpn I 
site in the 3' direction, it is preferable not to remove them because 
removal of additional nucleotides can disrupt the 3' LTR. 
Thereafter, one inserts a heterologous gene segment into the HIV segment 
wherein this heterologous gene segment corresponds to a sufficient number 
of nucleotides of the desired heterologous gene to express a functional 
product. This segment preferably contains the initiation and termination 
signals as well as the nucleotide sequences for the desired gene. 
Preferably, the heterologous gene segment is about 1200 nucleotides or 
less, more preferably, about 1000 nucleotides or less, and still more 
preferably, about 800 nucleotides or less. 
Preferably, one would use a heterologous gene that can be used as a marker. 
For example, the CAT gene corresponding to a gene that is less than about 
800 nucleotides. The entire coding sequence for this gene is located 
within a 750 nucleotide-long region. Furthermore, a simple sensitive assay 
for this enzyme activity is exists [Gorman, C. M., et al., Mol. & Cell. 
Biol. 2:1044-1051 (1982)] and antisera that recognizes the CAT protein is 
also readily available [Gorman, C. M. et al., Cell 42:519-526 (1985)]. 
Proviral plasmids incorporating a nucleotide sequence corresponding to the 
CAT gene can readily be made by inserting the nucleotide sequence between 
restriction enzyme cleavage sties, for example, the Xba I and Kpn I sites, 
using standard techniques. Other sites can also be used such for insertion 
of the heterologous gene segment, for example the Xba I site and the Xho I 
site within the 3' nef gene. 
The vector is then used to transfect a desired cell by standard techniques. 
For example, one can transfect a cell in vitro using, for example, the 
calcium phosphate coprecipitation technique. Alternatively, this vector 
can be used to transfect living cell in vivo. 
After transfection of the cells, either in vitro or in vivo, one would use 
standard techniques to detect the presence of the marker gene. For 
example, in the above-described instances where the vector contains a CAT 
gene, one merely measures for CAT activity. This technique would permit in 
vivo screening of the spread of the virus in various tissues. By 
determining which tissues show CAT activity, one can learn how the virus 
spreads. Additionally, one can also measure the quantity of CAT activity 
and thus determine the level of the virus in the various tissues. 
Using this vector to transfect cells in vitro can permit a very rapid 
system for screening drugs. With a sensitive marker like CAT, it is 
possible to detect the expression of the marker about one day after the 
cells have been transformed. Thus, using this vector, one can rapidly 
screen for drugs that affect the spread of the virus in infected cells and 
also for compounds that will affect the ability of the virus to infect a 
cell. For example, one can add a predetermined compound to a cell culture 
and thereafter try to transfect those cells with the vector of the present 
invention. Thereafter, one can measure the level of the marker in the cell 
to determine the effectiveness of a compound in preventing the spread of 
the virus. Alternatively, one can tranfect cells with the vector of the 
present invention, measure the level of activity of the marker and then 
add the predetermined compound to the cells. One then measures the 
activity of the marker to determine the effectiveness of the compound. 
By measuring the level of activity of the marker it is possible to 
determine, not only the effectiveness of the predetermined compound 
against the virus, but the degree of effectiveness of this compound. 
Controls can be run simultaneously or shortly after these test are run. 
Compounds that appear to show an effect against the virus in vitro can then 
be administered by standard techniques to one of the above-described in 
vivo systems to test the effectiveness of this compound in vivo. 
The present invention is further illustrated by the following examples. 
These examples are provided as an aid to understanding the invention and 
are not to be construed as a limitation thereof. 
EXAMPLES 
Cell Lines and Viruses 
The HIV proviral clone pHXBc2, as described in Fisher, A. G., et al., 
Nature 316:262-265 (1985) was used. The Jurkat cell line is a T4+ human 
malignant T-lymphoblastic line provided by the lab of Cox Terhorst. The 
cells were maintained at 37.degree. C. in a CO.sub.2 incubator in RPM1 
1640 medium supplemented with 10% of fetal bovine serum, glutamine, 
penicillin (1,000/ML) and streptomycin (100 .mu.g/ml). C8166 cells are an 
HTLV-1-transformed human lymphocyte line expressing very high levels of 
the T4 marker as described in Salahuddin, S. Z., et al., Virology 
129:51-64. The cells express no known products of HTLV-I except the tat 
protein. Cells were maintained under the same conditions as the Jurkat 
line. HeLa cells, a human cervical carcinoma line, were maintained under 
similar conditions except the medium used was Dulbecco's Modified Eagles 
Medium (DME) supplemented with 10% fetal bovine serum. 
DNA Transfections 
Jurkat cells were pelleted for 5 minutes at 1000 rpm, washed once with 
serum-free RPM1 medium, and then resuspended in fresh RPM1 medium at a 
density of 10.sup.7 cells/ml. One ml of cells were then added per 
transfection to tubes already containing the plasmid DNA in 2 ml of 
serum-free RPM1 plus 150.lambda. of a 5 mg/ml stock solution of DEAE 
dextran in 1M Tris-Cl pH 7.3. Tubes were then incubated at 37.degree. C. 
for one hour with occasional agitation. The cells were then pelleted, 
washed once with 10 ml of serum-free RPM1, and resuspended in 15 ml of 
RPMl plus 15% fetal bovine serum. All cells were subsequently given a 
complete medium change daily, throughout the course of the experiment. 
HeLa cells were seeded at a density of 10.sup.6 cells/100 mm plate the day 
before transfection. Transfection mixes contained the DNA in 1 ml of Hepes 
Transfection Buffer plus 0.11 ml of 1.25 CaCl.sub.2 added disguise with 
agitation. Transfection mixes were incubated for 30 minutes at room 
temperature, then added dropwise to the medium over the HeLa cells. 
Cells were then incubated for 24 hours. Afterwards the medium was removed 
and the cells were washed once with 7 ml of serum-free medium. The cells 
were then shocked by treatment with 2 ml of 10% DMSO. After 10 minutes the 
DMSO was aspirated and 10 ml of fresh medium were added to the plates. 
Cells were collected for assay 48 hours later. 
Cat Assays 
Cells were spun down, washed once with Phosphate Buffered Saline (PBS), and 
freeze/thawed three times in a small volume of 200 mM Tris, pH 7.5. 
Lysates were then analyzed for CAT activity in a standard assay mix 
containing .sup.14 C-chloramphenicol and Acetyl CoA as described in 
Sodroski, J. G., et al., Science 225:381-385 (1984). Percent conversion of 
chloramphenicol to the acetylated forms was determined by ascending 
thin-layer chromatography and liquid scintillation counting of the spots 
cut from the plate. 
Reverse Transcriptase (RT) Assays 
For each assay, 1 ml of culture medium from each sample was collected and 
centrifuged for one hour at 15,000.times.g to pellet virions. Pellets were 
resuspended in 10 .mu.l of 50 mM Tris HCl pH 7.5, 1 mM dithiothreitol, 
0.25 M KCl, and 20% glycerol. RT activity was assayed in 50.lambda. 
reaction mixes using oligo(dt)-poly (A) template-primer and magnesium 
cofactor, as described in Rho H. M., et al., Virology 112: 355-360 (1981). 
HIV p24 Assay 
1 ml of culture medium from each well was collected and centrifuged for 1 
hour at 15,000.times.g to pellet virions. Pellets were resuspended in 100 
.mu.l of assay buffer containing 0.5% Triton X-100, and assayed using a 
commercially prepared HIV p24 radioimmune assay kit (Lee, T. H., et al., 
Proc. Natl. Acad. Sci. USA. 81:3856-3860 (1984) according to manufacturers 
directions. Standards supplied with the kit were used to quantitate 
protein levels in the samples. 
Immunoprecipitation 
Cells were spun down, washed once with PBS, and resuspended in 2.5 ml of 
cysteine-free RPM1 plus 10% fetal bovine serum supplemented with 50 
.mu.ci/ml of .sup.35 S-cysteine. Cells were labelled overnight, then 
harvested, washed once with PBS, and lysed in 0.5 ml of 0.05 M tris, HCl 
pH 7.0, 0.15M NaCl, containing 1% Triton X-100, 1% sodium deoxycholate, 
and 0.1% SDS. Immunoprecipiation with AIDs patient antiserum and 
polyacrylamide gel electrophoresis were performed described in Lee, T. H., 
et al., PNAS 81, supra. 
Preparation of a Vector 
We used plasmid pHXBc2-Xba I, an infectious proviral clone derived from 
pHXBc2 [Fisher, A.G. et al, Nature 316, supra (1985)] which contains an 
artificially introduced restriction enzyme cleavage site near the 3' end 
of the gene encoding the envelope transmembrane protein [Lobel, L. I., 
PNAS 81:4149-4153. This mutation introduced a unique Xba I site, as well 
as a termination signal at a position 17 codons before the natural 
termination signal of the envelope glycoprotein (See FIG. 1). The presence 
of this linker insertion did not alter the kinetics of virus replication 
as compared to the pHXBc2 parent. 
Thereafter, sequences in this provirus between the unique XbaI site and a 
Kpn I site located 60 nucleotides 5' to the beginning of the 3' LTR were 
removed, resulting in a mutant provirus (pHXB-.DELTA.3'), which contains a 
deletion of 280 nucleotides including the 3' end of the env gene, the 3' 
nef initiation codon and 220 nucleotides of the 3' nef gene. Although the 
3' nef coding sequences extend beyond the Kpn I site in the 3' direction, 
it is preferable not to remove them because removal of additional 
nucleotides can disrupt the 3' LTR. 
A heterologous gene segment was then inserted. This segment preferably 
contains the initiation and termination signals as well as the nucleotide 
sequences for the desired gene. As shown, in FIG. 1 two DNA segments 
derived from the plasmid pBR322 were inserted in place of the deleted 
segment to produce the plasmids pHXB-.DELTA.3'-0.5 and pHXB-.DELTA.3'-1.2. 
pHXB-.DELTA.3' was constructed by deleting the region between a 12 base 
pair Xba I linker insertion into the 3' end of the env gene and a Kpn I 
site located within the 3' nef gene. pHXB-.DELTA.3'-0.5 and 
pHXB-.DELTA.3'-1.2 contain segments of pBR DNA inserted in place of the 
region deleted in pHXB-.DELTA.3'. The segment inserted in 
pHXB-.DELTA.3'-0.5 was derived from the 470 base pair pBR fragment located 
between the unique EcoR I and Sal I cleavage in that plasmid. The segment 
in pHXB-.DELTA.3'-1.2 was derived from a 1.17 kb fragment excised using 
the Sal I and Sca I sites. These proviral clones were then transfected 
into Jurkat cells and monitored daily for signs of HIV infection using the 
technique described above. Parameters checked included cell number, 
synctia formation and reverse transcriptase activity of the culture 
supernatants. See Table 1. 
Cells transfected as described above by pHXBc2-Xba I, which contained a 280 
nucleotide deletion of the env and 3' nef sequence as described above, 
replicated as well as that produced by the wild type provirus. Cells 
transfected with pHXB-.DELTA.3'-0.5, which in addition contained a 475 
nucleotide insertion for a net increase of 190 nucleotides also produced 
virus that replicated as well as the wild type. Cells transfected with 
pHXB-.DELTA.3'-1.2, which contained an insert of 1.17 kilobases for a net 
increase of about 890 base pair, produced virus which were much attenuated 
especially in initial replication. Transfection with this plasmid resulted 
in an initial appearance of only small numbers of synctia and very low 
levels of reverse transcriptase activity. Only at later times 
post-transfection (days 10-12) was there some increase in the number of 
synctia and the amount of reverse transcriptase activity in the 
pHXB-.DELTA.3'-1.2-transfected culture coincident with a decline in the 
number of viable cells. 
Filtered supernatant collected from the pHXB-.DELTA.3'-1.2-transfected 
culture at this time proved unable to initiate new cycles of infection 
when added to fresh uninfected Jurkat cells. Supernatants containing 
1000-2000 units of reverse transcriptase activity from cultures 
transfected with either wild type pHXBc2, pHXB-.DELTA.3', or 
pHXB-.DELTA.3'-0.5 plasmids were capable of initiating new rounds of 
infection in fresh Jurkat cells. 
A vector was then constructed using the gene for chloramphenicol 
acetyltransferase (CAT). This gene is available to the skilled artisan. 
The entire coding sequence for this gene is located within a 750 
nucleotide-long region. Moreover, a simple sensitive assay for its enzyme 
activity exists (Gorman, C. M., et al., Mol. and Cell. Biol. 2: 1044-1051 
(1982)) and antisera that recognize the CAT protein are available (Gorman, 
C. M., et al., Cell 42:519-526 (1985). 
Two vectors incorporating the coding sequences for the CAT gene were made. 
A segment of DNA approximately 800 nucleotides long that contained the 
entire coding sequence for the CAT gene as well as the translation 
initiation and termination signals was inserted into each provirus. For 
pHXB-CAT1 the CAT gene was inserted between the Xba I and Kpn I sites in 
place of the sequences removed in the deleted provirus pHXB-.DELTA.3' (See 
FIG. 1). For pHXB-CAT2 a similar insertion was made between the Xba I site 
and the Xho I site that is located within the 5' end of the 3' nef gene. 
In both constructions the CAT gene is located downstream from the natural 
splice acceptor used for production of the 3' nef protein (Muesing, M. A., 
et al., Nature 313:450-458 (1985). The plasmid pHXB-CAT1 has a net size 
increase of 570 bp over the wild type pHXBc2 proviral clone and in turn is 
110 bp smaller than pHXB-CAT2. Both vectors were transfected into Jurkat 
cells by the transfection technique described above, which were monitored 
daily thereafter for signs of HIV infection. 
FIG. 2 shows that the virus produced by the pHXB-CAT1 vector replicated 
almost as well as the wild-type virus. 
Jurkat cells were transfected on day 0 with over 10 micrograms of one of 
the proviral clones. The cells were monitored daily thereafter for signs 
of virus activity. Duplicate cultures were transfected with each plasmid 
to insure sufficient cells would be available for sampling. Media on the 
cells were changed daily and one-half of the cells from each culture was 
removed on day four to keep the cells for a longer time in log phase 
growth. FIG. 2A shows cell counts and uses the following symbols: 
.circle-solid.-mock transfection; .smallcircle.-pHXB-CAT2; 
.DELTA.-pHXBCAT1; .box-solid.-pHXB-.DELTA.3; and X-pHXBc2. FIG. 2B shows 
the reverse transcriptase activity where the symbols have the same meaning 
as above. Immunoprecipitation of protein is shown in FIG. 2C. 5 ml 
aliquots of each culture were collected 2, 5 and 9 days after transfection 
and labelled overnight with .sup.35 S-cysteine by standard techniques. 
Samples were then collected, detergent lysed and immunoprecipitated with 
AIDS patient antiserum using standard techniques. Lane A-mock 
transfection, Lane B-pHXB-CAT2, Lane C-pHXB-CAT1, Lane D-pHXB-v3' and Lane 
E-pHXBc2. 
The course of infection in cells transfected with pHXB-CAT1 appeared to lag 
behind that in the culture transfected with pHXBc2 by about 2-3 days, by 
whatever parameter was measured. However, by day 8-9 post-transfection the 
pHXB-CAT1 culture attained high levels of reverse transcriptase activity 
and dramatic cytopathic effect very similar to what was seen in the wild 
type culture. By contrast, although the culture transfected with pHXB-CAT2 
exhibited low levels of reverse transcriptase activity from three 
days-post-transfection onward and displayed significant syncytia 
formation, the infection did not progress normally and significant 
cytopathic effect was not observed as late as ten days post-transfection. 
Furthermore, filtered supernatant fluid taken from the culture transfected 
with pHXB-CAT2 at day ten did not produce indications of virus infection 
when applied to fresh Jurkat cells. Corresponding supernatants from 
cultures transfected with pHXBc2, pHXB-.DELTA.3', and pHXB-CAT1 all 
initiated new cycles of infection when cultivated with fresh cells, even 
when first diluted to compensate for differences in RT levels between the 
samples (data not shown). 
These observations are closely mirrored by the results of protein-labelling 
experiments described in FIG. 2C. HIV proteins are strongly evident in 
labelled aliquots from cultures transfected with either the wild-type or 
pHXB-.DELTA.3' constructs even when labelling was initiated only 48 hours 
post-transfection. The small difference seen in this particular experiment 
between the day 2 pHXB-.DELTA.3' and pHXBc2 lanes was not repeatable. 
Virus-specific proteins are only weakly evident in lanes representing day 
2 lysates from cells transfected with pHXB-CAT1 or pHXB-CAT2. However, by 
day 5 the virus-specific bands are stronger in the lysate from the 
pHXB-CAT1 culture and by day 9 the pHXB-CAT1 lane is virtually 
indistinguishable from the pHXBc2 and pHXB-.DELTA.3' lanes. By contrast, 
the day 5 lysate from the culture transfected with pHXB-CAT2 is not 
significantly different from the day 2 lysate. Only in the day 9 lysate 
can an increase in the intensity of the virus-specific bands in the 
pHXB-CAT2 culture be seen. Still at this time the amounts of viral 
proteins detected are well below those observed in the lysates from the 
other transfected cultures. 
To test for expression of the inserted CAT gene, aliquots of Jurkat cells 
transfected with pHXB-CAT1 were collected daily. Results of CAT assays for 
these samples are summarized in FIGS. 3A and 3B. 1 ml from the culture was 
collected daily. The cells were washed once with PBS and then resuspended 
in 150 .mu.l of 200 mM Tris at pH 7.5 for CAT assays. FIG. 3A is an actual 
autoradiogram of a CAT assay and represents conversions obtained using 
extract from Day 1 through Day 4 in the experiment. Each pair of spots in 
the autoradiogram represents conversion after 5 and 60 minute reaction 
times. The extracts from Day 3 post-transfection onward had to be assayed 
at 10 or 100-fold dilutions to maintain the conversion within the linear 
range of the assay. FIG. 3B summarizes the change in the level of CAT 
enzyme activity recorded over the entire course of the experiment. The 
scale used for the graph is percent conversion of .sup.14 
C-chloramphenicol per minute by .sup.70 .mu.l of 1.times.10 dilution of 
the original samples. 
CAT activity was easily detectable only one day post-transfection and 
quickly progressed to very high levels as the infection spread through the 
culture. The slight decline in the level of CAT expression at late times 
post-transfection presumably reflects the drastic loss in viability of the 
culture which was occurring by this time. 
Expression of the gag/pol and envelope proteins of HIV is known to require 
the presence of the rev gene product. Evidence also exists which suggests 
that the 3' nef gene product is made independently of rev. The mRNA for 
the 3' nef product splices out the known positive and negative cis-acting 
elements crucial to rev regulation. To test whether expression of CAT 
enzyme by pHXB-CAT1 is under rev control, we introduced a frame shift at 
the Bam HI site that eliminated the ability of the viral segment to 
produce a functional rev protein. The pHXB-CAT-1 and pHXB/BFS-CAT1 
plasmids were then co-transfected into plates containig 10.sup.6 HeLa 
cells with or without a rev expressor plasmid (pIIIexart) by the 
transfection method described above. HeLa cells, which do not express T4, 
were used so that re-infection of cells by the nondefective 
pHXB-CAT1-derived virus would not occur. See FIG. 4. 10 .mu.g of each 
proviral plasmid was used, plus 8 .mu.g of pIIIexart. Forty-eight hours 
post-transfection the cells were scraped into PBS, spun down, and 
resuspended in 150 .mu.l of 0.25 M Tris Cl pH 7.5 for CAT assays. The 
autoradiogram shows percent conversions obtained after 5 minutes (first 
lane of each pair) and 60 minutes (second lane of each pari) incubations 
using 70 .mu.l of each lysate. Control lysate was from transfection of a 
construction otherwise identical to pHXB-CAT1 in which the CAT gene was 
inserted in the reverse orientation (pHXB-CAT-anti). 
As shown in FIG. 4, CAT activity in cells transfected with pHXB/BFS-CAT1 
was similar to that seen in cells transfected with pHXB-CAT1. 
Co-transfection of HXB/BFS-CAT1 with a rev expressor plasmid, pIIIexart, 
resulted in about a 4-fold decrease in CAT activity. This is consistent 
with a reported decrease in the ratio of spliced to unspliced HIV mRNAs 
seen in the HIV mRNAs seen in the presence of rev. Therefore, expression 
of CAT in pHXB-CAT1 is not dependent on the presence of the HIV rev 
protein. 
An assay for screening anti-viral drugs using the pHXB-CAT1 vector was 
performed. Duplicate sets of C8166 cell cultures were infected with 
equivalent titers of virus stocks derived from either pHXBc2 or pHXB-CAT1. 
Different concentrations of either azidothymidine (AZT) [Mitsuya, H., et 
al, PNAS 82:7096-7100 (1985)], 2'3'-dideoxycytosine (ddC) [Mitsuya, H., et 
al, AIDS: Modern Concepts and Therapeutic Challenges pp. 303-333 (New 
York: Marcel Dekker) (1987)] or 2'3'-dideoxy-adenosine (ddA) [Mitsuya, H. 
et al, AIDS: supra]; three drugs known to inhibit HIV replicaiton, were 
added to the infected cultures. Cultures with no drug served as controls. 
As shown in FIG. 5, the marked decreases in viral protein expression 
observed in the presence of optimal doses of the different drugs 
correlates closely with changes in the level of CAT enzyme measured. The 
C8166 cells were set up in 24-well plates at a density of 2.times.10.sup.5 
cells/ml, 1 ml per well, in varying concentrations of drug or with no 
drug. Duplicate wells were set up for each drug concentration. 2,000 RT 
units of wild type pHXBc2-derived virus or 2,000 RT units of 
pHXB-CAT1-derived virus were added to each well. RT activity in the virus 
stocks was determined as described above. Cells were then incubated for 1 
week with a partial medium change on Day 4. Aliquots of the cells and 
media were collected and assayed on Day 7. 
FIG. 5A shows protein immunoprecipitations. Cells in wells infected with 
pHXBc2-derived virus were labelled overnight with .sup.35 S-cysteine. 
Samples were then collected, detergent lysed, and immunoprecipitated with 
AIDS patient antiserum. 
FIG. 5B shows the results of CAT assays. Cells in wells infected with 
pHXB-CAT1-derived virus were washed and collected in 85.mu.l of 0.25 M 
Tris Cl pH 7.5. The scale used in the graph represents percent conversion 
of .sup.14 C-chloramphenicol in 60 minutes by 70 .mu.l of each extract. 
The experiment was repeated using an equal mixture, as determined by 
measurement of cell-free reverse transcriptase activity, of the pHXBc2 and 
pHXB-CAT1 virus preparations. Under these conditions the presence of the 
replicating wild type virus did not inhibit the production of CAT enzyme 
by the pHXB-CAT1-derived virus. After one week the levels of CAT enzyme in 
cultures treated with increasing concentrations of AZT correlated closely 
with the total amounts of viral protein present in the cultures (data not 
shown). 
The results demonstrate that a foreign gene can be incorporated into an HIV 
vector and be successfully expressed without disrupting functions critical 
for virus replication and cytopathic effect. The major constraint appears 
to be size. The normal full-length HIV is apparently close to the maximum 
size permitted for efficient transmission, as proviruses with net size 
increases of 700 nucleotides or more compared to the wild type were unable 
to mount as successful infections following exposure of T-cells to 
supernatants from cells transfected with these plasmids. The results also 
show that the HIV-CAT1 virus can be used to measure the effect of 
anti-viral drugs. Use of this virus should permit a rapid, quantitative 
means of anti-viral drug activity. The results also show that a gene in 
the position of 3' nef can be expressed with absence of rev activity. 
The mutations in pHXB-.DELTA.3' introduces a premature termination codon 
into the env gene without appending additional amino acids. We conclude 
that the C-terminal 17 amino acids of the gp41 envelope glycoprotein are 
not required for normal replication and cytopathic activity of HIV in 
T-cells in culture. 
It is evident that those skilled in the art, given the benefit of the 
foregoing disclosure, may make numerous other uses and modifications 
thereof and departures from the specific embodiments described herein 
without departing from the inventive concepts, and the present invention 
is to be limited solely by the scope and spirit of the appended claims. 
TABLE 1 
__________________________________________________________________________ 
SYNCITIA CYTOPATHIC 
REVERSE CELL-FREE 
FORMATION 
EFFECT TRANSCRIPTASE 
TRANSMISSION 
PLASMID 
DAY 5 
DAY 12 
DAY 5 
DAY 12 
DAY 5 
DAY 12 
(2000 RT units) 
__________________________________________________________________________ 
pHXBc2 +++ + ++ +++ +++ +++ YES 
pHXB-.DELTA.3' 
+++ + ++ +++ +++ +++ YES 
pHXB-.DELTA.3'-0.5 
+++ + ++ +++ +++ +++ YES 
pHXB-.DELTA.3'-1.2 
+ ++ - ++ + ++ NO 
__________________________________________________________________________