Nov 18, 1991 - After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. ... They usually consist of two components: the retrovirus vector which ... After the gene transfer and expansion of infected cells in tissue ... been constructed in which most of the U3 region of the right. (3') LTR is ...
Vol. 66, No. 3
JOURNAL OF VIROLOGY, Mar. 1992, p. 1336-1343
0022-538X/92/031336-08$02.00/0 Copyright C) 1992, American Society for Microbiology
Unusually High Frequency of Reconstitution of Long Terminal Repeats in U3-Minus Retrovirus Vectors by DNA Recombination or Gene Conversion PAUL OLSON,' HOWARD M. TEMIN,2 AND RALPH DORNBURG1* Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854-5635,' and McArdle Laboratory
for Cancer Research, University of Wisconsin, Madison, Wisconsin 537062 Received 25 September 1991/Accepted 18 November 1991
Recently, we described a retrovirus vector system with which to study formation of cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 6:2328-2334, 1988; Mol. Cell. Biol. 8:64-72, 1990; J. Virol. 64:886-889, 1990). For these studies, retrovirus vectors were constructed in which the U3 region of the 3' long terminal repeat (LTR) was deleted. After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. However, the insertion of some additional sequences into such vectors promoted vector rearrangements with an efficiency greater than 95%. Such rearranged vectors behaved like vectors with two wild-type LTRs. Proviruses derived from such vectors were investigated by Southern blot analysis, polymerase chain reaction, and DNA sequencing. We found that the U3 region was reconstituted, resulting in vectors with LTRs like wild-type virus. The sequences that reconstituted the U3 region of the vector LTR were derived from LTR sequences present in the helper cell. Since no retroviral protein coding sequences were detected in infected target cells, recombination of vector sequences with coencapsidated helper cell sequences during reverse transcription seems very unlikely. Thus, it appears that the recombination (or gene conversion) events leading to a vector with reconstituted LTRs occurred at the DNA level. The high frequency of this recombination (or gene conversion) was dependent on internal vector sequences. Retrovirus vectors are the most efficient tools for introducing exogenous genes into vertebrate cells. They usually consist of two components: the retrovirus vector which contains the gene(s) of interest replacing retrovirus protein coding sequences and a helper cell which supplies the retrovirus proteins for the encapsidation of the vector genome into retrovirus particles without the production of replication-competent virus (7, 9, 32, 35, 42). In the last decade, several retrovirus vector systems, derived from chicken and murine retroviruses, have been developed for the expression of various genes (for reviews, see references 20, 33, and 40). Because of their efficient gene transfer properties, there is considerable interest in using retrovirus vectors in human somatic cell gene therapy (2, 8, 30, 34). In fact, recently the first clinical experiments have been initiated to apply retrovirus vector-mediated gene therapy to patients with adenosine deaminase deficiency. In addition, experiments have been started to treat cancer patients with malignant melanoma, a lethal skin cancer, with genetically manipulated tumor-infiltrating lymphocytes. In both applications, the gene of interest (adenosine deaminase gene or the gene coding for tumor necrosis factor, respectively) were introduced into cells of the patient in vitro by a retrovirus vector. After the gene transfer and expansion of infected cells in tissue culture, the cells were reinjected into the patients. The retrovirus vectors generally used today, including those used in the first gene therapy experiments, have full-length retrovirus long terminal repeats (LTRs) which contain the retrovirus promoter and enhancer. The genes of interest are expressed from either the LTR promoter or
*
Corresponding author. 1336
internal promoters, leading to the continuous expression of that gene. Consequently, the application of such vectors is limited in several ways. For example, regulated and controlled expression of the gene of interest is not possible when the gene is expressed from the LTR promoter. Furthermore, regulated expression of genes from internal promoters can be still affected adversely, since the retrovirus enhancer and promoter can interfere with internal promoters (18, 19). Thus, in certain cases in which regulated or inducible gene expression is required, vectors with intact LTRs are not likely to be useful. To circumvent problems with promoter interference and to enable expression of the gene of interest from a cell-typespecific promoter, retrovirus vectors of murine leukemia virus (MLV) and the avian spleen necrosis virus (SNV) have been constructed in which most of the U3 region of the right (3') LTR is deleted (3, 13, 45). U3 contains the retrovirus promoter and enhancer. After one round of replication, U3-deleted vectors no longer contain the retrovirus promoter and enhancer. Thus, gene expression is driven by internal promoters only (10) (Fig. 1). However, in the case of MLV-derived U3-minus vectors, the application of such vectors appears to be limited, since the titer of infectious particles was markedly reduced in comparison with results for vectors with two wild-type LTRs (45). In other experiments with MLV-based vectors, only the retrovirus enhancer was removed (16) or replaced by a cell-type-specific enhancer (8). However, the substitution of the enhancer did not result in cell-type-specific expression of the LTR. In the case of SNV U3-minus vectors, virus titers could be restored by the addition of a polyadenylation signal inserted downstream of the U5 region of the right LTR (13). We used U3-minus retrovirus SNV vectors to study the formation of processed pseudogenes by retroviruses (10, 11,
VOL. 66, 1992
RETROVIRUS VECTORS
STEP 1:
Transfect
plasmid into helper cells
LTR
hygre
1337
LTR
JD214HY LTR
__
E_ _= _ transrptio _
ter2 supF
hygro
TKproMLV-
ter
I transcription
TLTR Lter2 supF hygro TKpro
A.
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MLV-U
ter
pRD1 8a
Harvest virus from supernatant, infect fresh helper cells
STEP2:
U3pRD33
~~~~~~~u3-
-
ter2supF A
ter
1,.
reverse transcription, integration
E
HLV-U hygrtr2supF Tlpro neo @-; SEEK-M
hygro
TKproMLV-U3
transcription
STEP3: HARVEST VIRUS, INFECT FRESH Di 7 CELLS. TRANSFER OF HYGROMYCIN RESISTANCE ?
FIG. 1. Experimental protocol. Retrovirus helper cells were transfected with plasmid DNA (step 1). DNA in the helper cell is the same as in the plasmid. RNAs transcribed from the LTR promoter contain all cis-acting sequences required for normal retrovirus replication. Virus was harvested from confluent cultures, and fresh helper cells were infected (step 2). This infection results in the formation of a U3-minus provirus. Thus, after one round of replication, the hygro gene (25) is expressed from the internal promoters only (promoter of the MLV U3 region and promoter of the herpes simplex virus thymidine kinase gene [TKpro]). The further transfer of Hygror from the infected step 2 helper cells was tested by infecting fresh D17 cells with virus harvested from such helper cells followed by selection for Hygror (step 3). Abbreviations: U3-, deletion of the U3 region of the right LTR with the exception of the attachment site; ter, polyadenylation sequence of simian virus 40; ter2, polyadenylation sequence of the herpes simplex virus thymidine kinase gene; supF, suppressor tRNA gene of Escherichia coli. Plasmid sequences (which abut the retrovirus vector sequences) are not shown.
12) (Fig. 1). In the course of these studies, many vectors were constructed to study the effect of defined sequences on the process of retroposition of nonretrovirus RNAs. Howseveral of these vectors did not behave in the predicted Proviruses that contained reconstituted (wild-type) LTRs were formed. This reconstitution of the U3 region appears to occur at the DNA level, using helper cell sequences as templates.
ever, way.
MATERIALS AND METHODS Nomenclature. All vectors were derived from SNV, an avian reticuloendotheliosis virus. Plasmid constructs are indicated by the letter p (e.g., pRD18) to distinguish them from virus (e.g., RD18). The hygromycin B phosphotransferase gene (isolated from pLg89 [25]) is referred as hygro. Hygror (hygromycin resistance) refers to the phenotype. The neomycin (G418) resistance gene is referred as neo (28). Plasmid constructions. All plasmids (Fig. 2) were constructed by standard cloning procedures (31). Protocols of constructions are available upon request. All vectors were derived from SNV. pJD214HY is a vector that contains all regulatory sequences required for normal retrovirus replication. The hygro gene is expressed from the LTR promoter. In all pRD vectors, most of the U3 region of the right LTR was deleted. The selectable gene (hygro) and all other
~~~~U3-
LTR
ter2 supF hygro
pRD48
TlCpro 11LV-U3Z
_f
ter
_
FIG. 2. Retrovirus vector constructs. All vectors were derived from SNV and contain SNV LTRs and all other cis-acting sequences for retrovirus replication. pJD214HY is a retrovirus vector that contains full-length LTRs. The hygro gene is expressed from the LTR promoter. In all pRD vectors, the U3 region of the right LTR was deleted and substituted with an XhoI linker (see also Fig. 1). Abbreviations: neo, neomycin resistance gene (28); MLV-E, encapsidation sequences of SNV and MLV, respectively. All other abbreviations are the same as in the legend to Fig. 1.
nonretrovirus sequences (e.g., internal promoter[s] and internal polyadenylation signal) were inserted in the opposite orientation to vector transcription. pRD18 was described previously (10). pRD33 and pRD48 were derived from pRD18. In pRD33, the neo gene was inserted between the two internal promoters of pRD18 (BamHI site). pRD48 is a retrovirus vector that contains an additional encapsidation sequence from MLV. The MLV encapsidation sequence (PstI-BalI fragment, map units 0.563 to 0.747 [1, 37]) was inserted between the two internal promoters (BamHI site) of pRD18. The orientation of the additional encapsidation sequence was the same as that of the hygro gene. The vector frames of pRD33 and pRD48 are identical to that of pRD18; i.e., except for these inserts, their sequences are identical. Cells. D17 dog cells and D17 C3A2 dog helper cells were grown as previously described (10). The helper cells (C3A2) were derived from D17 cells and supply all retrovirus proteins essential for virus replication without production of replication-competent helper virus (42). Selection of Hygror cell colonies were performed in medium containing hygromycin B at 80 jig/ml. Transfections and infections. Transfections were performed by the Polybrene-dimethyl sulfoxide method (29). Virus titers were determined on D17 cells as described previously (10) and are expressed as CFU per milliliter of tissue culture supernatant medium. In all experiments, virus was harvested from cells after selection for drug resistance. DNA isolation and Southern blots. Chromosomal DNAs were isolated by proteinase K digestion and phenol-chloroform extractions (31). Southern blots were performed under standard conditions (38). PCR, cloning of PCR-amplified fragments, and DNA sequencing. Polymerase chain reaction (PCR) amplification was performed by using the Repliprime kit (NEN DuPont) according to the manufacturer's instructions. Triton X-100 was added to a concentration of 0.07% to reduce nonspecific amplification. Amplified fragments were made blunt ended with Klenow polymerase and resolved on a 1% agarose gel. Individual bands were excised, GeneClean (Bio-101) puri-
1338
OLSON ET AL.
J. VIROL.
TABLE 1. Transfer of Hygror from step 1 and 2 helper cells' Virus titer (CFU/ml) Provirus
JD214HY RD18 RD33 RD48
Step 2 (C3A2 cells)
Step 3 (D17 cells)
5 x 105 2 x 102 3 x 103 1 x 104
107 1 7 x 105 106
" Virus was harvested from step 2 helper cells infected with vector virus as indicated. Fresh D17 cells were infected. Titers (expressed as Hygror CFU per milliliter of tissue culture supernatant medium) at step 3 (see text) were determined as described previously (10). Values are averages from five individual cell clones.
fied, and cloned into the HinclI site of Bluescript II SK+ (Stratagene). Double-stranded templates were sequenced by the dideoxy chain termination method (Sequenase V2.0 sequencing kit; U.S. Biochemical). RESULTS Experimental protocol. Recently, we developed a retrovirus vector system to study formation of processed pseudogenes by retroviral proteins (10-12). The experimental protocol of these experiments is outlined in Fig. 1. Plasmids were transfected into retroviral helper cells (step 1). RNAs were transcribed from the LTR as well as from the internal promoter. Transfected cells were selected for Hygror and passaged. RNA transcribed from the LTR promoter had all cis-acting sequences required for retrovirus replication and was encapsidated into virus particles supplied by the helper cell. Virus was harvested from confluent Hygror cultures, and fresh helper cells were infected. In the case of several vectors (e.g., pRD18; Fig. 1), this infection resulted in the formation of U3-minus proviruses. RNA was transcribed from the internal promoter only (Fig. 1, step 2 [10]). Hygror helper cell colonies were isolated to give step 2 helper cell lines. Virus was harvested from confluent cultures, and fresh D17 target cells were infected (step 3). In the case of vectors containing a tandem promoter (e.g., pRD18; Fig. 1), Hygror resistance was transferred to the fresh D17 target cells at a frequency about 7 orders of magnitude less than that of transfer of Hygror by a vector containing all cis-acting sequences required for retrovirus replication (Table 1). We have shown that this gene transfer resulted from the formation of cDNA genes: mRNAs starting from the MLV U3 promoter were reverse transcribed and inserted into the genome of the infected target cells to form functional cDNA genes. The hygro gene was expressed from the second internal promoter. However, all of the cDNA genes investigated did not correspond to a full-length mRNA and lacked the hallmarks of naturally occurring processed pseudogenes (10, 12). Unusual vector behavior. In the course of further studies, we constructed a series of vectors with additional sequences to test the effect of such sequences on cDNA gene formation. Many of these vectors did not form predicted U3-minus proviruses. Some of these vectors are shown in Fig. 2. In this report, we describe the frequent reconstitution of the U3 region of two of these vectors, pRD33 and pRD48. pRD33 is a retrovirus vector with two internal genes transcribed from the internal promoters. pRD48 is a vector containing the encapsidation sequences (Psi) of MLV between the two internal promoters (see also Materials and Methods). (This
vector was made to test the effect of Psi on the efficiency of cDNA gene formation [11].) pRD33 and pRD48 were used in the experimental system outlined in Fig. 1. After transfection of the plasmids and selection for Hygror, virus was harvested from confluent cell cultures and fresh helper cells were infected. One day after infection, cells were selected for Hygror and virus titers were determined (Table 1, step 2). Single-cell colonies were isolated, and cell lines were established from such step 2 helper cell clones. Virus was harvested from confluent cultures, fresh D17 cells were infected, and the virus titers were determined (Table 1, step 3). In the case of pRD18, Hygror was passaged to the (step 3) target cells at a very low efficiency. As reported earlier, this transfer is the result of cDNA gene formation from RNAs transcribed from the internal promoters (10, 12). However, in the case of pRD33 and pRD48, Hygror resistance was passaged with efficiencies similar to the efficiency of Hygror transfer by the JD214HY vector (Fig. 2), which contains two wild-type LTRs (Table 1). Furthermore, if RD33- and RD48-infected cells (step 3 cells) were superinfected with reticuloendotheliosis virus (REV-A), an avian retrovirus closely related to SNV, Hygror was efficiently transferred to fresh D17 target cells. The efficiency of this transfer (virus titers of 106 for both RD33 and RD48) was identical to that of the control vector, JD214HY, which contains two wild-type LTRs. No Hygror was transferred from RD18-infected step 3 cells. In these experiments, 10 plates of D17 cells were infected with a total of 2 ml of tissue culture supernatant medium harvested from RD18 step 3 cells. Such cells were shown to contain cDNA genes. These cDNA genes were derived from RNAs without retroviral cis-acting sequences (10). Southern analysis of recombinant proviruses. The high efficiency of Hygror transfer from step 2 helper cell lines to D17 cells with the RD33 and RD48 vectors suggested that recombination or gene conversion had occurred with these vectors. To test this further, Southern blot analyses with chromosomal DNAs isolated from RD33 and RD48 step 2 helper cell lines were performed. In all of our U3-minus vectors, the U3 region of the right LTR was replaced with an 8-bp linker containing the restriction enzyme cleavage site for XhoI. Thus, after one round of normal retrovirus replication, XhoI sites are present in both U3-minus LTRs. This marker provides an excellent tool with which to investigate correct provirus formation by monitoring XhoI-digested chromosomal DNAs (10, 13). None of the cell clones derived from RD33 and RD48 revealed a band of the expected size (Fig. 3). Bands were observed in the high-molecular-weight fraction of the gel. These data indicate that the XhoI site was not duplicated during replication. Thus, RD33- and RD48infected cells do not contain the expected provirus. On the basis of the experiments described above, we hypothesized that recombination (or gene conversion) led to a recombinant vector with two wild-type-like LTRs. To test this hypothesis further and to determine the step at which these rearrangements occurred, we modified our experimental protocol. Virus was harvested from helper cells transfected with pRD33 or pRD48. Fresh D17 cells were infected and selected for Hygror. The number of Hygror colonies was 1 order of magnitude higher than that of RD18 from which these vectors were derived (data not shown). D17 cells do not contain a helper virus activity which would lead to the further passage of SNV-derived vectors. Thus, these infected D17 cells contain proviruses that went through only one round of retroviral replication. First, we tested whether Hygror was transferred by a virus
VOL. 66, 1992
RETROVIRUS VECTORS Xhol
4.1 kbp e
RD18
a b (: (1
c.
r~~~~~~~J
Xhol
RD33
Xhol
oTK prO?-ILV-
1339
f
kbp
5.4 kbp
Xhol
tt e r2 supF hygro TKpro
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.t
Xhol
4.5 kbp
ter2supF hygro TK-pro
Xhol M
'1. 1
Vul
R D48 MLV-E
RD18
___s
RD33
3.0
I
RD48
2.0
-
1.6
i:
.I I:
-4-
FIG. 3. Southern blot analysis of step 2 RD18, RD33, and RD48 proviruses. Hypothetical proviruses are shown at the top (see legend to Fig. 1 for abbreviations). Chromosomal DNAs of step 2 helper cell lines were isolated, digested with XhoI, and subjected to Southern blot analysis. Filters were hybridized with a hygro-specific probe. From left to right: DNAs isolated from three different RD18 cell clones, five different RD33 cell clones, and five different RD48-derived helper cells clones. Arrows indicate the position of bands if correct proviruses were formed.
arose by sequences or
-
.4-
I
that
4,1-
recombination of the vector with helper virus by the occasional production of replicationcompetent virus (which has been reported for C3A2 helper cells [26]). In either of these events, we would expect to detect SNV protein coding sequences in infected D17 cells. To test for the presence of SNV protein coding sequences, chromosomal DNA was extracted from a pool of about 20 to 50 Hygror cell colonies. The DNA was double digested with HindlIl and Sacl (which cut several times within the viral genome) and subjected to Southern blot analysis. The filter was hybridized with DNA specific for SNV protein coding sequences (Fig. 4 shows hybridization with an envelopespecific probe). No SNV protein coding sequences were detected in D17 cells infected with RD33 or RD48 (Fig. 4, lanes a and b, respectively). Several bands were detected in the parental helper cells which contain several copies of the helper cell genome (lanes c and d). Further Southern blot analysis of the D17 cell DNAs digested with various enzymes and hybridized with a hygro-specific probe revealed that the internal structure of the vector had not been changed (lanes e and f and data not shown). These data show that the transfer of Hygror did not occur by a recombinant virus containing SNV protein coding sequences. Furthermore, these data indicate that only the deleted U3 region of the vector has been repaired. Analysis of reconstituted LTRs by PCR and DNA sequencing. To investigate reconstituted LTRs at the molecular level, we analyzed the right LTR of proviruses formed after one round of retrovirus replication. Such LTRs were amplified by PCR, cloned, and sequenced. Choosing one primer
1.0
FIG. 4. Southern blot analysis of D17 cells infected with RD33 and RD48 as well of parental helper cell DNAs. Chromosomal DNAs were digested with HindIll and Sacl. Lanes: a and b, DNAs isolated from step 2 D17 cells; c and d, DNA from step 1 helper cells (the filter was hybridized with an SNV envelope-specific probe); e and f, step 2 D17 cell DNA infected with RD33 and RD48, respectively. The DNA was digested with HindIIl plus Ball. The filter was hybridized with a hygro-specific probe.
specific for sequences in the polypurine tract permitted selective amplification of the 3' (right) LTR. The other primer was specific for the 3' end of the U5 region. A total of 14 clones positive for LTR sequences were sequenced and compared with a wild-type SNV LTR. Eight clones were derived from RD48 provirus, and six clones were derived from RD33 provirus. Ten of the fourteen clones sequenced had full-length LTRs. Four of these were derived from RD33, and six were derived from RD48 (Fig. 5 and data not shown). One clone (derived from RD48) had a correctly sized U3-minus LTR; three other clones (derived from RD33) revealed substantial deletions of U3, R, and U5 (Fig. 6). (i) Proviruses with fully reconstituted LTRs. As outlined above, 10 clones obtained in our experiments had fully reconstituted LTRs (Fig. 5 and data not shown). Since the sequences determined differed markedly from the published SNV LTR sequence (44), we used the sequence of pJD214HY as a reference (top line of Fig. 5). (This sequence was determined in parallel experiments.) All pRD vectors were derived from pJD214HY and therefore should contain LTRs identical to those of pJD214HY. In two clones with reconstituted LTRs, the U3 region was derived from REV-A LTR sequences (Fig. 5 shows the sequence of one of these clones, termed 48.E, in comparison with the published sequence of REV-A [bottom line]). (C3A2 helper cells contain chimeric viral genomes of SNV and REV-A, two closely related retroviruses [42; see also Materials and Methods].) In all other fully reconstituted LTRs sequenced, the U3 regions were derived from SNV LTRs. All reconstituted LTRs had several point mutations and small deletions in comparison with the vector LTR.
1340
T
J. VIROL.
OLSON ET AL. J021 4HY 33. R3 33 .A?
40.15 48.16
t8.12 *68 .E
GtGGGRGGGRGTCTGGGGGGTGTGCTGGCTCGCTPRCTGCTRTRTTRGCTTCTGTRCCCRTGCTGCTTGCCCTGGCCRCTRRCCGCCRT
TTRGC
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33. R3 33 .R7 40.15 48 .16 40.12
.................................................................................................... .................................................................................................... .................................................................................................... .................................................................................................... ....................................................................................................
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CGCTTGTCGRRRTCGGCGTCRTTTCTCGGCRTCGRGRGCRRGCTCRTARRRCCRTRRRffGGRRTGTGTRTTGRRGGCRRGCRTCRGRCCRCTTGCGCCRTC
33. R3 33. R7 §@ .15 48.16 40 .12 W6E RE" -
.................................................................................................... .................................................................................................... .................................................................................................... .................................................................................................... ....................................................................................................
J0214tHY 33. R3 33 .R? 48.15 48 .16 40.12 t6WE RE" -
----------------------------
TCG..G .G R......G. ...G.................RGG G............... TTG............. R... ............
-------------------------------
-----
CRRTCRCGRRCGRRCRCGRGRTCGGRCTRTCRTRCTGRGCCRRTGGTTGTRRRGGGCRGRTGCTRCTCTCCRRTGRGGGRRRRTGTCRTGT RRCRCCCTG ............................... ............ ........................
.......................................
............................................
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33.AR? 48.15 16.16 48.12
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JD214HY 33.A3 33 .R? 48 .15 48.16 40.12 48.sE REU-R
J021 4HY 33.R3
33.R7 46.15 48.16 46.12 W6.E REU-R
R U3 TRRGCTGTRRGCGGCTRTRTRRGCCGGGTRCRTCTC TTGCTCGGGGTCGCCGTCCTRCRCRTTGTTGTTGTGRCGTGCGGCCCRGRTTCGRRTCTGTRRT ........
..........
--..--........ R ............
.................................................... ........
..........
......................
--. R G TG.C A.TT . .............T. C..A ...G
... .--C
....
...
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R | US RRRRCTTTT TTTTTTCTGRRTCCTCRGRTTGGCRGTGRGRGGRGRTTTTtGTTCGTGGTGTTGGCTCGCCGRCTGtGGTGGGGCRGGGRTC .___ GGRCT ...........................
..... ..
Z........................................... -RCC ...... ;RTC ---------------------RT T. RC
................................................................... .......
....
..........................
.
-.........
.
..
...... RC
...
...................... -................................
....G ....................................... G .T ............. G T--RGG G. C C....GC T ....
...
......
GRRTCGTRGTRTTCGGTRC ........................ ........................ ........................ ........................ ........................ ........................ ............
.........
T
FIG. 5. DNA sequences of reconstituted LTRs of RD33 and RD48. The sequence of a wild-type LTR (derived from pJD214HY; Fig. 2) is shown at the top. The sequence of an REV-A LTR is shown at the bottom. Only the differences compared with the SNV LTR are indicated. A point indicates sequence identity; a dash indicates a deletion. The sequences of full-length reconstituted LTRs of RD33 and RD48 are shown in the middle. Sequences designated 33.A3 and 33.A7 were derived from RD33. The other sequences were derived from RD48. The box R r o 46.15ates ad11 -e th site cloning rposes.
VOL. 66, 1992
RETROVIRUS VECTORS 11
Hyp.Seq 33.1 33.12
21
31
41
OTOGGROGGG CTCGRGG CGGGGTCGCCGTCCTRCRCRTTGTTGTTOTGR
33.2 48 .F
........... tctgggg gaatag-------------------------..........:tctgggggootogt------------------------....
..
...
....
...
.
_--___--________-___-___-___-___-___-
.....T....... ................................. TG GGT GGG
Hyp. Seq
CG TGCG GCC CRGR TTC GRRTCTGTRRTRRRRCTTTTTTTT TTC TGRR TCC
33.1 33.12 33.2 48 .F
__- -___ --_ -___ --__ -__ --___ --_ --_____
Hyp.Seq
TC RGRT TGG CRGT GRG RGG RGRT TTT GTTCGT GGTG TTGG CTC GCCG RCT
33.1 33.12 33.2
*8 .F
.... ....
--
----
---
__- -___
--_-
...
.... ----
-___
--__
-__
--___
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___
____
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.. .. .. .. .. .. .. .. .. .. ...
------
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_,,
____
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. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .
Hyp .Seq
GGGTGGGCG CRGGGRT C GR TC CG GRC TGRRTC CGTRGTRC TTC GGTRCRRC
33.1 33.12
.................
....
............
33.2
48.F
................
_ ___
_
_
..............
____. _ _
_
_
_
FIG. 6. DNA sequences of correctly formed proviruses of RD48 and proviruses derived from RD33 and RD48 that are partially reconstituted but contain extensive deletions of U3, R, and U5. The sequence of a hypothetical U3-minus LTR is shown at the top. The XhoI linker is marked with a box (top). Small letters indicate sequences present in the wild-type SNV LTR. One clone revealed an 8-bp insertion (bottom line) 1 bp in front of the XhoI linker. The box (bottom) indicates a BamHl site that was filled in in all pRD constructs by Klenow polymerase and then ligated. A dash indicates the deletion of one base.
(ii) Proviruses with deleted LTRs. Besides the 10 clones with full-length LTRs, we also obtained one clone from RD48 with a correctly sized U3-minus LTR (Fig. 6, bottom line). Correctly sized U3-minus proviruses were not detected by Southern analysis in 10 of 10 individual RD48 cell clones investigated or in the DNA isolated from a pool of about 50 cell colonies (Fig. 3 and data not shown). These data show that, in the case of RD48, almost all of the input vectors (most probably greater than 95%) were converted back to wild type. No correctly formed U3-minus LTRs were found in RD33 clones. However, in three other clones derived from RD33, LTRs that contained some sequences of the original U3 region replacing the XhoI linker were found. However, most of the U3 region, R, and some U5 sequences were missing (Fig. 6, clones 33.1, 33.12, and 33.2, respectively). In addition to the reconstitution of the U3 region, we also found significant changes in the U5 region. During the construction of the pRD vectors, we destroyed a BamHI site in the U5 region of the 5' (left) LTR for cloning purposes. This was done by filling in the sticky ends after BamHI digestion with Klenow polymerase and ligating the ends. However, in all clones sequenced, these modified sequences reverted to wild type or had other mutations near the original BamHI site (Fig. 5 and 6, boxed sequences).
DISCUSSION established a retrovirus vector system with which to study formation of processed pseudogenes (Fig. 1) (10-12). In the course of these investigations, retrovirus Recently,
we
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vectors were constructed in which the U3 region of the right LTR was deleted and replaced with a XhoI linker (e.g., pRD18; Fig. 2). As the result of normal retrovirus replication, a provirus with two U3-minus LTRs which contained an XhoI site in both LTRs was formed after one round of virus replication. In this report, we describe two other vector constructs (pRD33 and pRD48) which did not behave in the predicted way. These vectors were derived from pRD18 by the insertion of additional sequences (the neo gene or the MLV encapsidation sequence). The insertion of these sequences promoted rearrangements of the vector at unusually high frequencies. The U3 region was rearranged in more than 95% of the proviruses. Most of the vector proviruses contained two LTRs like wild-type virus. The reconstitution of the U3 region could theoretically occur either at the DNA level by DNA recombination or gene conversion or at the RNA level during reverse transcription (template switching with coencapsidated helper virus RNA). Recently, it was shown that template switches during reverse transcription occur at fairly high frequencies. It was estimated that 3 to 5% of proviruses had undergone at least one template switch (6, 27, 39). DNA recombination has been proposed as a mechanism for the transduction of cellular genes by retroviruses (22, 23). In the case of recombination with coencapsidated helper cell sequences during reverse transcription, we expect to find retrovirus protein coding sequences in infected target cells. We did not detect retrovirus protein coding sequences in the infected target cells (Fig. 4). Thus, it appears that the rearrangements that led to the reconstitution of the LTRs took place at the DNA level in the helper cell after transfection. It may be argued that occasionally coencapsidated readthrough transcripts from the internal vector promoters that contain LTR sequences could act as primers restoring U3. However, the efficiency of encapsidation of such RNAs (without encapsidation sequence as in the case of pRD33) is at least 4 orders of magnitude less than of retrovirus RNA (10). Thus, in this case, we would expect that only 0.1% of proviruses are rearranged. However, the frequency of rearrangements was much higher (greater than 95%). Furthermore, the virus titer at step 2 was increased in the case of pRD33 and pRD48 in comparison with pRD18 (Table 1). In addition, reconstitution of U3 was also observed in vectors that do not produce minus-strand RNAs (pJD220SVHY [12a, 14]). These data further indicate that the rearrangements occurred in the helper cell. It may also be argued that DNA recombination between two plasmid DNAs during transfection gave rise to abnormal vectors with wild-type LTRs. However, two recovered vector proviruses contained U3 regions that were derived from REV-A LTRs which are only in the helper cell genome. Furthermore, almost all recovered vectors contained BamHI sequences which are also only in the helper cells. These data show that the recombination events occurred between chromosomal DNA and the vector DNAs. However, it is unclear whether these rearrangements occurred before or after the vector plasmid had integrated into the genome. Some recovered LTRs had extensive deletions comprising some sequences of U3, the complete R region, and some sequences of U5 (Fig. 6). In two cases, the XhoI linker present in the plasmid construct was lost and replaced with short sequences of the original U3 region (indicated by small letters in Fig. 6). It may be speculated that those LTRs represent products of incomplete recombinations or gene conversions. It is interesting to note that R is believed to be essential for retrovirus replication because it mediates trans-
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fer of the first-strand cDNA to the 3' end of the second RNA molecule (41). In our vector constructs, the polyadenylation sequence of simian virus 40 was inserted in the BamHI site of U5. It was shown that RNAs of such vectors are preferentially polyadenylated downstream of this poly(A) site (13). As a consequence, such vector transcripts also contain some U5 sequences at the 3' end. If the R region at the 3' end was missing as a result of incomplete DNA rearrangements, the downstream U5 region may have served as a recipient for the hybridization of the transferred first-strand cDNA. Recently, the recombination of retroviral vectors, e.g., the reconstitution of the U3-minus LTR, was investigated in C3A2 and in DSN helper cells (14). C3A2 helper cells contain multiple copies of the SNV and REV-A wild-type LTRs. In DSN cells, gag-pol and env proteins are expressed from the cytomegalovirus immediate-early gene promoter or the promoter of Rous sarcoma virus, respectively. Thus, they are free of SNV or REV-A LTRs. The frequency of LTR restoration in another U3-minus vector (pJD220SVHY) was reduced in DSN cells by 1 to 2 orders of magnitude (14). These experiments establish that the presence of wild-type LTRs in the helper cell contributes to rearrangements of the vector. However, they also indicate that the U3 region of the left LTR may also serve as a template for reconstitution, although with much lower frequency. In addition to the reconstitution of the U3 region of the 3' LTR, we observed another rearrangement in the U5 region that led to the reconstitution of a BamHI site which had been destroyed for cloning purposes (Fig. 5 and 6, boxed sequences). Since the BamHI sites of both the 3' and 5' LTRs are destroyed in all pRD vector constructs (the BamHI site of the 3' LTR was used for the insertion of the simian virus 40 polyadenylation signal) (Fig. 2; see Materials and Methods), it can be concluded that helper cell sequences served as template for this second rearrangement. These data further show that the rearrangements described here took place at the DNA level. It is interesting to note that in other experiments with other vector constructs from avian leukosis virus containing mutations at a similar position, revertants to wild type were observed with high frequencies when wild-type sequences were present in the helper cell (5, 30a). The LTRs of reconstituted vectors contained many point mutations and small deletions. The frequency of these mutations is much higher than the frequency of background mutations introduced by the PCR technology (17). Thus, these mutations may result from errors made by reverse transcriptase. Alternatively, these mutations could have accumulated in the LTRs present in the C3A2 helper cells before they were used as the template for reconstitution of the vector. C3A2 helper cells have been kept in tissue culture for almost 10 years. Thus, no definite conclusion can be made about the origin of these mutations. However, investigation of mutation rates of the SNV reverse transcriptase by others makes the latter explanation more likely than frequent errors introduced by the retrovirus polymerase (15, 36). These data further indicate that the reconstituted U3 region of the vector was derived from helper cell sequences. Our data suggest that the insertion of some sequences in a stable retrovirus vector can result in an unstable vector construct. Some of our vectors, such as pRD18, were repeatedly tested in several transfection/infection experiments. No recombinant vectors were observed. However, it appears that the insertion of some sequences between the two internal promoters triggered rearrangements that led to the reconstitution of the U3 region at unusually high frequen-
cies. For example, in the case of pRD33, no correctly sized proviruses were detected by PCR in a pool of about 50 step 2 infected cell clones, indicating that rearrangements of the U3 region occurred in all transfected cells. Recently, we described another retrovirus vector to test the effect of an encapsidation sequence on the efficiency of cDNA gene formation after retrovirus infection (termed pRD47 [11]). pRD47 is identical to pRD48 except that the thymidine kinase promoter of pRD48 is replaced with the simian virus 40 early gene promoter. In the case of pRD48, no correctly sized provirus was detected in 10 of 10 cell clones infected with RD48 (Fig. 3 and data not shown). However, in the case of RD47, 50% of infected step 2 cell lines (5 of 10) contained correctly sized proviruses and behaved as expected (11). The other RD47-derived cell clones with incorrectly sized provirus gave results similar to those obtained with RD48 (data not shown). This result shows that even subtle differences like the choice of the internal promoter influenced the frequency of the rearrangement of the vector. In summary, we have shown that the U3 region of U3-minus retrovirus vectors can be reconstituted in helper cells. This rearrangement appears to occur at the DNA level. However, we cannot distinguish at this point whether it was the result of homologous recombination or gene conversion. The frequency of these rearrangement events was unusually high (over 95%) in the case of some vectors. It appears to depend on internal vector sequences. However, the properties and the mechanism that trigger these events are not understood. ACKNOWLEDGMENTS We thank S. Hinz, J. Couch, and W. Sheay for technical assistance. We thank Joe Dougherty, Celine Gelinas, and Arnold Rabson for helpful comments on the manuscript. The research in H. M. Temin's laboratory was supported by Public Health Research grants CA-22443 and CA-07175 from the National Cancer Institute. H.M.T. is an American Cancer Society Research Professor. REFERENCES 1. Adam, M. A., and D. Miller. 1988. Identification of a signal in a murine retrovirus that is sufficient for packaging of non-retroviral RNA into virions. J. Virol. 62:3802-3813. 2. Anderson, W. F. 1984. Prospects for human gene therapy. Science 226:401-409. 3. Anderson, W. F., E. F. Wagner, and E. Gilboa. 1986. Selfinactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. Acad. Sci. USA
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