Complementation of a Vesicular Stomatitis Virus ... - ScienceDirect

VIROLOGY

178,

373

-383

(I 990)

Complementation Protein Expressed

of a Vesicular Stomatitis Virus Glycoprotein G Mutant with Wild-Type from either a Bovine Papilloma Virus or a Vaccinia Virus Vector System ELLIOT J. LEFKOWITZ,’

Department

of Mtcrobiology,

ASIT K. PATTNAIK,

The University Received

March

of Alabama

AND

at Blrmmgham,

8, 1990; accepted

May

L. ANDREW

BALL

Birmingham.

Alabama

35294

25, 1990

Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins. The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV). This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected. These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (tsO45) at the permissive and nonpermissive temperatures. A lOO-fold increase in tsO45 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells. These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase. T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells. This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals. VSV G protein expressed by this system was able to complement fsO45 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system. When calcium phosphatemediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained. Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells. At the nonpermissive temperature, yields of temperature-sensitive virus were within IO-fold of the yields obtained at the permissive temperature. Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype. This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication. Q 1990 Academic Press, Inc

INTRODUCTION

whtch would not be possible using conventronal approaches. In contrast to the replication of most DNA genomes, the replication of RNA involves no proofreading capability. The high error rate of the RNA-dependent RNA polymerase of RNA viruses, therefore, results in the production of high numbers of viral mutants (reviewed in Steinhauer and Holland, 1987). The frequency of appearance of monoclonal antibody-resistant mutants (MARMs), which may reflect genome changes at a s/ngle nucleotide site, is reported to be on the order of 1 Om5to 1 Oe4 for the hemagglutinin of Sendai virus, the surface glycoprotein molecule (G) of vesicular stomatitis virus (VSV), and the hemagglutrnin of influenza A virus (Portner et a/., 1980). Virus mutation frequencies as determined by the appearance of MARMs may, in many cases, have been underestimated due to phenotypic mixing with wild-type protein pools (Holland eta/., 1989; Valcarcel and Ortin, 1989). These results suggest that the actual frequency of appearance of MARMs is much closer to the 10 -4 to 1Om3range rem ported for the appearance of single nucleotide changes as detected by direct sequencing (Steinhauer

The ability to generate and propagate negativestrand RNA vrruses which contain lethal mutations would provide a powerful tool for further study of the function and interaction of viral proteins in viva. For negative-strand RNA viruses, it is difficult to directly engineer mutations into the viral genome as is possible for many DNA viruses and positive-strand RNA viruses. Genetic studies of negative-strand RNA viruses has been limited to the isolation of naturally occurring mutants which by necessity have to exhibit conditional activity in order to replicate. Characterization of the phenotypes of these mutants, along with the expression of individual wild-type and mutant genes engineered into eukaryotic expression vectors, has allowed significant progress in understanding the structure and function of the gene products of these viruses (reviewed in Pringle, 1987). In this report we describe a system which may allow the generation and propagation of viral mutants

’ To whom

requests

for reprints

should

be addressed 373

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$3.00

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et a/., 1989a). This high error rate presents

LEFKOWITZ,

PATTNAIK.

a problem for RNA viruses which, in order to remain infectious, must maintain a functional complement of proteins. For an RNA virus such as VSV with a genome of approximately 11,000 bases, and considering the fact that the RNA polymerase has an error rate of approximately 1 Op4 misincorporations per nucleotide per round of replication, it has been estimated that following one round of replication most of the replicated genomes differ from each other by at least one nucleotide. The wild-type nucleotide sequence of such a virus should be thought of as a consensus sequence with any individual virus genome differing from the consensus at one or more positions (Steinhauer et al., 1989b). While the polymerase error rate may allow for rapid genome evolution and virus adaptation to different environments, the wild-type consensus sequence is presumably normally maintained by selection pressure for functional viral proteins. Indeed, the importance of selection pressure during viral replication is evident from the observation that the frequency with which temperature-sensitive mutants of VSV arise is 1 to 2% (Flamand, 1980). This high mutation frequency is understandable when considering the single nucleotide substitution rate of 1 Oe4 to 1Om3, and the size of the VSV genome. In addition, these results do not take into account nontemperature-sensitive mutations, and the production of lethal mutants. It is likely that the actual frequency of mutants that result in an altered phenotype may be much higher. It seems likely that selection pressure for functional viral proteins should be exerted on each gene in a more-or-less independent manner. Thus, if it were possible to relieve the selection pressure on the product of a single viral gene, we would expect to see an increased frequency of mutations accumulating only in that gene. Examination of such an experimental system should provide information on the relationship between polymerase error rates and mutation rates, and on the contribution of selection pressure in the maintenance of wild-type consensus sequences. To relieve selection pressure on a single viral protein, we have examined two complementation systems in which the G protein of VSV is provided by a genetic source other than the VSV genome itself: a bovine papilloma virus (BPV) expression vector (Sarver eta/., 1981) and the vaccinia virus (VV)-T7 RNA polymerase (VV/T7 pol) expression system (Fuerst et al., 1986). Evaluation of the ability of VSV G protein expressed from these two systems to complement temperature-sensitive mutants of VSV was undertaken as a prerequisite for an examination of the mutability of the G protein gene during passage of wild-type VSV through the complementing systems.

AND

BALL

The BPV system is based upon transcription of the gene of interest from a BPV vector which contains SV40 early region expression signals and the required BPV transforming sequences (Sarver et a/., 1981). These BPV-based plasmids are transfected into cells which subsequently become transformed as a result of expressing the appropriate BPV proteins. The BPV plasmid is then maintained in the transformed cells as a multicopyextrachromosomal episome. Expression of the protein of interest is directed by SV40 promoter and termination signals. This system has the advantage of establishing a cell line which constitutively produces the cloned protein. The VVfT7 pol system is dependent on the expression of bacteriophage T7 RNA polymerase from cells infected with a vaccinia virus recombinant containing the T7 RNA polymerase gene (Fuerst et al., 1986). Following infection, the cells are transfected with a plasmid that contains the gene of interest cloned between T7 promoter and terminator signals. This allows transcription of high levels of mRNA when theT7 RNA polymerase produced by the VV recombinant initiates transcription at the T7 promoter on the transfected plasmid. This system has allowed the production of very high levels of protein from VV recombinant-infected, plasmid-transfected cells, though protein production eventually stops as the VV infection kills the cells. During the course of our investigation, other groups have shown that the replication of temperature-sensitive mutants of VSV G protein (Whitt et a/., 1989) and VSV M protein (Li et a/., 1988) can be complemented using this system. We chose to express G protein of VSV from these two expression systems due to its wide use in the past for similar complementation experiments. VSV G protein has been previously expressed from SV40-based vectors (Rose and Bergmann, 1982); adenovirus vectors (Schneider et a/., 1989); VV-based recombinants (Mackett et a/., 1985); BPV vectors (Florkiewicz et al., 1983; Florkiewicz and Rose, 1984); and VVfT7 pol vectors (Keil and Wagner, 1989; Whitt et a/., 1989). VSV G protein is required for the attachment of the virion to the host cell (Bishop et al., 1975) and is also involved in penetration and uncoating of the virion in the host cell (Matlin et al., 1982). In addition, the G protein is probably involved in the assembly and budding of newly formed virions through the cell membrane (Metsikko and Simons, 1986). A temperature-sensitive mutant in VSV G protein, ts045 (Flamand, 1970; Flamand and Pringle, 1971) contains a single amino change in the hydrophobic domain of the protein (Gallione and Rose, 1985). At the nonpermissive temperature, this mutant G protein does not mature in a normal manner, and is not transported to the cell surface (Bergmann

COMPLEMENTATION

OF A VSV

et a/., 1981). This results in the production of “bald,” noninfectious virions with no VSV G protein present on the exterior of the viral envelope (Schnitzeret a/., 1979). A truncated fragment of the mutant G protein is inserted into the plasma membrane of infected cells and probably plays a role in budding of thevirions (Metsikko and Simons, 1986). As an initial step in the project to examine the hypothesis that complementation of VSV G protein during replication of wild-type virus will allow the virus G coding sequence to drift and therefore allow us to isolate and propagate G protein mutants, we evaluated the utility of the BPV and VV/T7 pol based expression systems for complementation of tsO45 replication. MATERIALS

AND

METHODS

Cells and viruses Mouse Cl 27 cells (Lowy et a/., 1978), human HeLa T4 cells (Maddon et al., 1988), and African green monkey BSC-40 cells (Hruby et a/., 1979) were maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Stocks of wild-type VSV (Indiana serotype, San Juan strain) and the temperature-sensitive G glycoprotein mutant ~~045 were grown in BSC-40 cells from plaquepurified stocks. The wild-type stock was prepared at 37”, while the ts045 stock was prepared at 33.5”. The recombinant vaccinia virus which expresses the T7 bacteriophage RNA polymerase (vTF7-3) was kindly provided by Dr. Bernard Moss of the National Institutes of Health (Fuerst et al., 1986). Stocks of this recombinant virus were prepared in BSC-40 cells. Virus assays All virus plaque and yield assays were carried out in 35-mm six-well multiwell plates using BSC-40 cells plated at a density of 1O6 cells per well. The plates were incubated at a permissive temperature of 33.5” or a nonpermissive temperature of 38.5 or 39” as indicated. For VSV plaque assays in the presence of VV, 25 pg/ ml of cytosine-@-p-arabinofuranoside was added to the agarose overlay to inhibit VV plaque formation. Plasmids Plasmid pBPV-VSVGAS was kindly provided by Dr. John K. Rose of Yale University. This plasmid is essentially the same as pSVBPVG (Florkiewicz et al., 1983) with the exception that a deletion had been constructed in the SV40 small t intron to remove a splice site which might have allowed utilization of a cryptic splice site in the VSV G gene (Puddington et al., 1987).

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MUTANT

375

This plasmid contains the 69% transforming region of BPV joined to pSV2, a pBR322 vector containing the SV40 small t antigen expression signals (Mulligan and Berg, 1980). The VSV G gene (Indiana serotype) which had been cloned between the t antigen transcription initiation and termination signals is expressed under control of the SV40 early promoter. In order to remove the pBR322 sequences this plasmid was digested with Xhol and religated prior to transfection. Plasmids pdBPV-1 and pdBPV-VD5 were transfected into cells, and transformed cell clones which contained these plasmids were used in complementation experiments as non-G protein-producing cell lines. Plasmid pdBPV-1 contains the complete BPV genome and the prokaryotic plasmid pML2d which is a “poisonminus” derivative of pBR322 (Sarver et al., 1982). Plasmid pdBPVVD5 was constructed by cloning the SV40 small t antigen expression signals from pSV2 into pdBPV-1. The VV gene coded for by open reading frame 5 of the VV Hindlll D region (Evans and Traktman, 1987) was cloned between the transcription Initiation and termination signals. Plasmid pTF-G was kindly provided by Dr. M. Abdul Jabbar of the University of Californra at Los Angeles. pTF-G contains the VSV G gene (Indiana serotype) cloned into the BarnHI site of pTF7-5 (Fuerst et al., 1986) between the T7 promoter and transcriptional terminator. Transcription of the VSV G coding sequence is dependent on the presence of T7 RNA polymerase. Isolation of BPV-transformed expressing VSV G protein

cells

Mouse Cl 27 cells were transfected with either pdBPV-1, pdBPV-VD5, or pBPV-VSVGAS using calcium phosphate precipitation as described by Sarver et a/., (1981). Approximately 2 weeks following transfection, transformed cell clones were picked and screened for the presence of the VSV G gene by Southern blot analysis of total cell DNA using a VSV G probe (data not shown). Positive clones were then recloned at least one more time prior to being used for analysis of VSV G protein expression and complementation of a VSV G mutant. The G protein producing clones, VSV G. l-l, G. l-6, and G. l-1 2, are all subclones of the same initial pBPV-VSVGAS-transformed clone. G protein-producing clones GA1 , GA2, GA5, and GA6 are subclones of G. l-1. BPV is a transformed clone isolated from cells transfected with pdBPV-1. This clone contains the complete BPV genome but IS missing the pML2d sequences present in the original plasmid. The VD5 clones are all separately derived clones of cells transformed with pdBPVVD5.

376

Expression the V/l/T7

LEFKOWITZ,

of VSV G protein pol system

PA-NAIK,

using

HeLa T4 or BSC-40 cells were used to express G protein using the VVfT7 pol system. The cells were plated at a confluency of approximately 90% (10” cells per well) and infected with vTF7-3 at a multiplicity of infection (m.0.i.) of 5 plaque forming units (PFU) per cell. Following virus adsorption, the cells were transfected with 15 pg of either pTF-G or, as a non-G proteinproducing control, salmon sperm DNA, using the calcium phosphate precipitation method, or using the cationic lipid, lipofectin (Felgner et a/., 1987). Calcium phosphate-DNA precipitates were prepared as described by Sarver et al. (1981). At 4 hr following the addition of the calcium phosphate precipitate, the cell monolayers were shocked by exposing them to 10% glycerol, washed, overlayed with medium, and incubated at 37”. Lipofectin (BRL, Gaithersburg, MD) was used at a final concentration of 20 pg per 10” cells using the manufacturer’s published procedures. Following 4 hr of incubation, the medium was adjusted to 2% FBS and incubation continued at 37”. lmmunoprecipitation In order to detect expression of VSV G protein, cells were labeled with [35S]methionine, and then cytoplasmic extracts were prepared as described by Florkiewicz et a/. (1983). VSV-specific proteins were immunoprecipitated with a rabbit polyclonal antibody raised against detergent-disrupted virions of purified VSV. This antibody was kindly provided by Dr. Richard Compans of the University of Alabama. Either goat anti-rabbit immunoglobulin immunobeads (Bio-Rad Laboratories, Richmond, CA) or Pansorbin (Calbiochem, La Jolla, CA) were used to adsorb and pellet the immune complexes. The samples were then electrophoresed on SDS-l 0% polyacrylamide gels (SDS-PAGE) using the buffer system described by Laemmli (1970). Following electrophoresis, the gels were fixed, washed, and treated with the fluor sodium salicylate (Chamberlain, 1979). The gels were then dried and exposed to X-ray film. lmmunoblot

analysis

Unlabeled cytoplasmic extracts were prepared as for immunoprecipitation with the exception that no ,&ME was included in the electrophoresis loading buffer. Following electrophoresis, the proteins were electroblotted to lmmobilon PVDF transfer membrane (Millipore Corp., Bedford, MA). VSV Gprotein was specifically detected using the rabbit anti-VSV polyclonal antibody.

AND

BALL

Bound antibodies were detected with a rabbit antimouse antibody conjugated to horseradish peroxidase (ICN Immunobiologicals, Costa Mesa, CA) as described by Samson et al. (1986). Flow cytometry Flow cytometry analysis was used to detect the expression of G protein on the surface of cells as described by Chan et al. (1988). Cells were scraped into PBS containing 5 mM EDTA, washed, and incubated with a mouse monoclonal antibody to G protein kindly provided by Dr. Richard Compans. The cells were washed, and then incubated with a phycoerythrin (PE)conjugated goat anti-mouse antibody (BioMeda, Foster City, CA). Following incubation the cells were washed, fixed, and analyzed for PE fluorescence on a Becton-Dickinson FACStar flow cytometer.

RESULTS Synthesis

of VSV G protein

in BPV-transformed

cells

In order to obtain a cell line which would constitutively produce VSV G protein, mouse Cl 27 cells were transfected with pBPV-VSVGAS and transformed cell clones were picked and screened by hybridization for the presence of intact plasmid DNA (data not shown). Cell clones which contained intact pBPV-VSVGAS DNA also showed the expected VSV G-specific mRNA band when analyzed by Northern blot hybridization (data not shown). Cells transformed with pBPV-VSVGAS exhibited the same growth characteristics as the control pdBPV-1 -transformed cells. The production of VSV G protein was detected directly by immunoprecipitation. Monolayers of BPVtransformed cells were labeled with [35S]methionine and proteins from cytoplasmic extracts were immunoprecipitated with a polyclonal antibody to VSV and analyzed by SDS-PAGE. The fluorogram of the gel is shown in Fig. 1. G protein was produced by all clones isolated from cells transformed with pBPV-VSVGAS, while cells transformed with pdBPV-1 produced no G protein. No major differences were seen in the capacity of different pBPV-VSVGAS-transformed cell clones to produce G protein. Cell surface expression of VSV G protein was quantitated by flow cytometry (data not shown). Cell clones which were transformed with pBPV-VSVGAS showed positive fluorescence while control, non-G protein-producing cell lines showed only background levels of fluorescence. For G protein-producing clone GA2, 95% of the cells were positive for G protein-specific fluorescence.

COMPLEMENTATION

I

Anti-VSV ““l”fWA~d

OF A VSV

Antibody IVSV

NiNS

M

FIG. 1. lmmunopreciprtation of VSV G protern from BPV-transformed clones. Transformed cell clones were grown rn the presence of 100 &/ml of [%]methronrne for 3.5 hr at 37”. Clones BPV and G. l-1 were also labeled from 4 to 5 hr followrng infection with wrldtype VSV. VSVspecrfrc proteins from cytoplasmtc extracts of these labeled cells were rmmunoprectprtated with a rabbit anti-VSV polyclonal antibody, separated on SDSPAGE, and fluorographed. The VSV-infected cells show bands correspondrng to VSV G, N, and NS (whrch comtgrate under these condrtrons), and M protetns.

Complementation of ~~045 replication transformed, VSV G protein-producing

GLYCOPROTEIN

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377

and VD5.8 giving the lowest levels of replication (data not shown). The capacity of any particular clone to replicate VSV was consistent in repeated experiments over the course of several months, but after prolonged passage in monolayer cultures, all clones showed a decrease in permissiveness for VSV replication (see below). This variability was not dependent on G protein expression since both G protein-expressing and nonexpressing cells showed clone to clone variation in permissiveness for VSV replication. At 38.5” the significant levels of virus obtained in VSV G protein producing cells over that in nonproducing cells indicated that the wild-type G protein was functional and could complement the temperature-sensitive defect of the G protein from ~045. The non-G protein-producing clones BPV, VD5.1, and VD5.8 produced 200 to 400 PFU of virus per 10” cells, while the G protein producing clones G.l-1, GA1 , and GA2 produced between 4000 and 20,000 PFU of virus per 1O6 cells. This represents a complementation efficiency of 10. to lOO-fold. Not all G proteln-producing clones were able to complement ~045. As can be seen in Fig. 2, clones G.l-6 and G. l-1 2 did not show any significant virus production at the nonpermissive temperature over that seen for the nonproducing clones. This is in

by BPVcells

Monolayers of VSV G protein-producing cells or nonproducing cells were infected with tsO45 and incubated at the permissive temperature of 33.5”, or the nonpermissive temperature of 38.5”. Following incubation, cell supernatants were harvested and VSV titers were determined by plaque assay at both the permissive and nonpermissive temperatures. Virus titers at the nonpermissive temperature gave an indication of the extent of revertant virus which may have been produced during the course of replication. Previous reports have indicated a reversion frequency for ~~045 of approximately 1Oe5 to 1Od4 (Flamand, 1980; Gallione and Rose, 1985) Titers of revertant virus produced by all of the transformed clones were less than 100 PFU/ 1O6 cells, which was consistent with the previously reported reversion frequencies. The results of a typical complementation experiment are shown in Fig. 2. Virus yield at 33.5” gives an indication of the permissiveness of each cell clone for its capacity to replicate VSV. As much as a 1OO-fold variation in the capacity of different cell clones to replicate tsO45 was seen. Replication of wild-type VSV also showed a similar variability from clone to clone with clones such as G.l-1, GAl, and VD5.1 giving the highest levels of VSV production, and clones such as G.l-6, G.l-12,

n

33.5”

m

38.5”

E G Cell

_ G l-12

Line

FIG. 2. Complementatron of a VSV G temperature-sensrtrve mutant In VSV G protein producing, BPV-transformed, cell clones. For a vrrus yield assay, confluent cell monolayers were Infected with rsO45 at a multrplrcrty of 50 plaque forming untts (PFU) of vrrus per cell. After 24 hr at the Indicated temperature, newly replrcated vrrus was harvested and then quantrtated In a plaque assay on BSC-40 cells. The temper ature-sensrtrve phenotype of the vrrus produced by the different cell clones was determined by plaqurng the virus at either the permrssrve (33.5”) or nonpermrssive (38.5”) temperatures. In these experiments. levels of revertant virus produced at 38.5” were less than 100 PFU for all of the cell clones. G. I-6, G. 1-l 2. Cell clones transformed wrth pBPV-VSVGX Isolated along with G 1 1

378

LEFKOWITZ. PATTNAIK. AND BALL

spite of the fact that these cell clones produced levels of G protein which were comparable to those produced by G.l-1, GA1 , and GA2 (data not shown). The most reasonable explanation for these data is that clones G.l-6 and G.l-12 are two of the least permissive clones for VSV replication, and this reduced ability to support VSV replication at the permissive temperature prevents effective complementation at the nonpermissive temperature. This was a consistent observation among many of the cell clones which were screened for complementation. Effective complementation of tsO45 at the nonpermissive temperature required not only the production of wild-type VSV G protein by the cell clone but also a capacity to efficiently replicate VSV at the permissive temperature. The levels of virus production obtained during complementation never approached virus titers obtained at the permissive temperature. At best, VSV G proteinproducing cells at the nonpermissive temperature gave titers of ~045 which were about lOOO-fold less than titers obtained at the permissive temperature. A 1O,OOO-fold difference was more common. In addition to these low levels of complementation, a problem arose as the transformed cells were continually passed as monolayer cultures. With time, even cell clones which previously produced respectable VSV titers at the permissive temperature became increasingly refractory to VSV replication. This decrease in permissiveness for VSV replication was accompanied by a reduced capacity to complement 1~045 replication at the nonpermissive temperature (data not shown). For these reasons an alternate system for expression of VSV G protein was evaluated for its capacity to produce functional protein and complement ~045 replication.

Analysis of VSV G protein production from the VV/T7 pol system Cells infected with the VV recombinant vTF7-3 produce bacteriophage T7 RNA polymerase in the cytoplasm. If these cells are then transfected with a plasmid (pTF-G) containing the VSV G gene cloned between T7 RNA polymerase promoter and terminator signals, a mRNA coding for VSV G is transcribed. Following translation of the VSV G message, functional VSV G protein should be detectable on the cell surface. lmmunoprecipitation of proteins from pulse-labeled cells was used in order to examine the rate of synthesis of G protein at various times after infection. As shown in the left panel of Fig. 3, when HeLa T4 cells were transfected with pTF-G using lipofectin, equivalent rates of G protein synthesis were observed at 6 and 12 hr post-transfection, while at 24 hr, the rate of G protein synthesis was significantly reduced. The reduction in

lmmunoprecipitation

G

Western Blot

G NINS

NINS

FIG. 3. lmmunoprecipitation and immunoblot analysis of VSV G protein expressed from the VVTT7 pol system using lipofectin-mediated transfection of pTF-G. Immunoprecipitation: HeLa T4 cells infected with vTF7-3 and transfected with pTF-G using the lipofectin procedure were labeled for 1 hr at 37” with 100 hCi/ml of [%methionine at the indicated times of 6, 12, or 24 hr post-transfection. Following labeling, cytoplasmic extracts were prepared and VSVspecific proteins were immunoprecipitated with a rabbit anti-VSV polyclonal antibody. The immunoprecipitated proteins were separated by SDS-PAGE, and fluorographed. Proteins from control, salmon sperm DNA-transfected cells were labeled for 1 hr at 12 hr post-transfection and treated as indicated above (control DNA lane). Proteins from VSV-infected cells isolated at 6 hr postinfection (VSV lane) or mock-infected cells (control lane) are also shown. Immunoblot (Western blot): HeLa T4 cell cytoplasmic extracts were prepared from vTF7-3 infected and pTF-G Iipofectin-transfected cells which had been incubated at 37” for either 6, 12, or 24 hr following transfection. Extracts from salmon sperm DNA transfected cells (control DNA lane) were prepared at 12 hr post-transfection. Extracts from either VSV-infected (VSV lane) or mock-infected cells (control lane) were prepared at 6 hr postinfection. The cytoplasmic extracts were electrophoresed by SDS-PAGE and then electroblotted to Immobilon PVDF membrane. VSV-specific proteins were detected with a rabbit anti-VSV polyclonal antibody and a horseradish peroxidaseconjugated secondary antibody.

G protein synthesis at 24 hr was probably due to cytopathology caused by the VV infection which was clearly visible at this time. Total accumulation of G protein in these cells was determined by immunoblot (Western blot) analysis. Proteins present in cytoplasmic extracts of vTF7-3 infected, pTF-G-transfected HeLa T4 cells were separated by SDS-PAGE, and electroblotted to an lmmobilon PVDF membrane. The membranes were then immunostained with the rabbit anti-VSV G polyclonal antibody. As shown in the right panel of Fig. 3, G protein was seen at 6 hr post-lipofectin transfection of pTF-G, and accumulated to high levels at 12 hr post-transfection. Further accumulation was seen up to 24 hr indicating that while the rate of protein synthesis may have significantly decreased by this time point, high levels of G protein remained associated with the cell.

COMPLEMENTATION

OF A VSV GLYCOPROTEIN

379

MUTANT

G protein expression was unaffected by temperature of incubation, and equivalent amounts of protein were seen when comparing expression in BSC-40 and HeLa T4 cells (data not shown). In subsequent experiments, HeLa T4 cells were used to express G protein since a less severe cytopathic effect was seen with these cells upon VV infection in comparison with BSC-40 cells. This allowed higher levels of complementation of tsO45 to be observed in HeLa T4 cells than in BSC-40 cells (data not shown). Increasing the m.o.i. of VV above 5 PFU/cell did not significantly increase G protein expression (data not shown). Detection of VSV G protein expressed VVA7 pol system on the cell surface using flow cytometry analysis

from the

In initial experiments, calcium phosphate precipitation was used to transfect the plasmid pTF-G into the vTF7-3-infected cells. A recent publication by Whitt et al. (1989) has suggested that transfection mediated by cationic lipids such as lipofectin is much more efficient in introducing DNA into VV-infected cells for expression by T7 RNA polymerase. Therefore, we wanted to compare VSV G protein production in cells transfected with DNA mediated by either calcium phosphate or lipofectin. In order to quantitate the levels of VSV G protein appearing on the cell surface, flow cytometry of intact, antibody-labeled cells was employed. In Fig. 4, the appearance of G protein on the cell surface at different times post-transfection is displayed. When using lipofectin transfection, G protein continued to accumulate on the cell surface for at least 24 hr post-transfection, while for calcium phosphate, expression peaked at 12 hr. In addition, lipofectin transfection resulted in up to five-fold greater levels of G protein expression in comparison to calcium phosphate on a per cell basis. With lipofectin transfection, G protein was expressed in over 90% of the cells, while only 50-600/o of the cells expressed detectable levels of G protein when calcium phosphate was used. When cells were infected with wild-type VSV at a m.o.i. of 10 PFU/cell, slightly higher levels of G expression were seen at 6 hr than for the VVfT7 pol system. By 12 hrfollowing VSV infection, the cell monolayer was mostly destroyed and flow cytometry analysis was impossible. It is, therefore, difficult to determine whether lipofectin transfection actually results in significantly greater levels of G protein production at 12 and 24 hr in comparison with VSV infection. It is clear that expression of G protein mediated by infection with vTF7-3 and lipofectin transfection of pTFG produced levels of G protein which were comparable to the levels of protein seen in an actual VSV infection,

0

6

Hours

12

16

1 24

post Transfection

FIG. 4. Flow cytometry of cells expressrng VSV G protean from the VVTT7 pol system. HeLa T4 cells, followrng rnfectron with vTF7-3, were transfected wrth either pTF-G or salmon sperm DNA usrng lipofectrn or calcium phosphate prectprtation and incubated at 37”. Cells were harvested at 6, 12, and 24 hr post-transfection. VSV G protern expressron at the cell surface was detected by Indirect rmmunofluorescence usrng a mouse ant&VSV G protein monoclonal antrbody and a phycoerythrrn-conjugated secondary antrbody Cells Infected with VSV at a m.o.r. of 10 PFWcell and Isolated at 6 hr postrnfectron were treated In the same manner. Cell surface fluorescence was detected In a flow cytometer and the average levels of VSV G protein expressed per cell are shown as mean positive fluorescence intensity. The background fluorescence of control, salmon sperm DNAtransfected cells was subtracted from the fluorescence intensity of pTF-G-transfected cells. The efftcrency of G protern expression from a population of cells is shown as the percentage of cells exhrbrttng posltrve fluorescence.

and that most of the cells in a population were able to show these high levels of protein expression. For these reasons, it was hoped that efficient complementation of ~045 at the nonpermissive temperature could be obtained using the VV/T7 pol-lipofectin system. Complementation of tsO45 by cells expressing G protein from the VV/T7 pol system

VSV

HeLa T4 cells were infected with vTF7-3 and transfected with pTF-G using either lipofectin- or calcium phosphate-mediated transfection. These cells were then superinfected with tsO45 at different times posttransfection at a m.o.i. of 10 PFU/cell, and then incubated at 33.5 or 39”. Virus yields were determined in a plaque assay at both 33.5 and 39” in order to quantitate temperature-sensitive and wild-type (revertant) progeny.

380

LEFKOWITZ, Lipofectin Transfection

73

PATTNAIK,

Calcium Phosphate Transfection 39”

33.5”

AND

BALL

levels of complemented virus than if VSV G protein was produced only after tsO45 replication had begun. Up to 50% of the background virus yields obtained at the nonpermissive temperature from cells not expressing VSV G protein represented revertant virus as assayed by its capacity to plaque at the nonpermissive temperature. This level of revertant virus is what might be expected to be produced when considering the levels of complemented virus produced in the pTF-Gtransfected cells at the nonpermissive temperature, and the reported reversion frequency of ~045 of approximately 1 Od5 to 1O-4. Levels of revertant virus produced in pTF-G-transfected cells were essentially the same as those seen in the control, salmon sperm DNAtransfected cells. Thermostability

6

Time

of lnfectlon

with

ts045

(Hours

post

of complemented

virus

8

transfection)

FIG. 5. Complementation of tsO45 in cells expressing VSV G protein from the VViT7 pol system. HeLa T4 cells were infected with vTF7-3 at a m.o.i. of 5 PFU/cell and then transfected using either the calcium phosphate or lipofectin procedure with 15 wg of pTF-G or salmon sperm DNA. At the indicated time post-transfection, the cells were superinfected wrth tsO45 at a m.o.i. of 10 PFUkell for 1 hr at 31”, washed, and then incubated at either 33.5 or 39” as indicated. Cell supernatants were harvested at 14 hr post-fs045 infection. Virus yields were determrned by plaque assay on BSC40 cells at 33.5”. pTF-G, cells transfected with pTF-G; control, cells transfected with salmon sperm DNA.

As is shown in Fig. 5, at the nonpermissive temperature, virus yields of between 1O4 and 1 O6 PFU/l O6 cells were obtained when the cells were transfected with pTF-G using calcium phosphate. This represents a complementation efficiency of lo- to 1500-fold. Transfection of pTF-G using lipofectin resulted in virus yields of 1 O6 to 10’ PFU/l O6 cells at the nonpermissive temperature, representing a complementation efficiency of 2000- to 15,000-fold. These virus yields were within 50-fold of the virus yields produced at the permissive temperature. In other experiments using lipofectin, over 10’ PFU/lO” cells were produced at the nonpermissive temperature. This was within 1 O-fold of the virus yield obtained at the permissive temperature (data not shown). In these experiments, the timing of tsO45 superinfection had only marginal effects on the complementation efficiency. In other experiments, if the cells were infected with ~045 2 hr prior to infection with vTF7-3, only a 50-fold complementation efficiency was obtained (data not shown). Allowing the accumulation of VSV G protein in the cells prior to rsO45 infection, therefore, resulted in the production of much higher

Virus produced in pTF-G-transfected cells at the nonpermissive temperature should remain genotypitally temperature-sensitive for growth while having a wild-type phenotype for G protein due to the incorporation of wild-type G protein in the viral envelope. Virions of tsO45 grown at the permissive temperature are heat-labile and lose infectivity when exposed to high temperatures (LaFay, 1974). Therefore, it was possible to determine the source of G protein in the virion envelope by measuring the heat lability of the virus. Temperature-labile virus would contain G protein produced from the mutant tsO45 G coding sequence. Temperature-stable virions would contain wild-type G protein. Figure 6 shows the results of a plaque assay at 33.5” of different virus stocks which were treated at 40” for different periods of time. The original ~045 stock lost essentially all of its infectivity after 2 hr of heat treatment, while the wild-type stock retained significant levels of infectivity. Virus isolated from &045-superinfected cells which had been transfected with salmon sperm DNA and incubated at 33.5” (control 33.5“ curve) was also heat-labile indicating that passage of tsO45 through non-G protein-producing cells did not alter the G protein phenotype. Infection of pTF-G-transfected cells with tsO45 and incubation at 39” (pTF-G 39” curve) produced virus which exhibited a temperature-stable phenotype similar to wild-type virus. The slope of the heat lability curve for this virus was the same as for the wild-type virus stock with only a small downward shift revealing a slightly greater heat-labile phenotype than for wild-type virions. This observation is consistent with the explanation that the complemented virus population contained wild-type VSV G protein in the viral envelope, but at slightly reduced levels when compared with the wild-type virions. If ~045 was passed through pTF-G-transfected cells at 33.5“

COMPLEMENTATION