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Virology 357 (2007) 41–53 www.elsevier.com/locate/yviro

Matrix protein of VSV New Jersey serotype containing methionine to arginine substitutions at positions 48 and 51 allows near-normal host cell gene expression ⁎ Gyoung Nyoun Kim, C. Yong Kang

Department of Microbiology and Immunology, The University of Western Ontario, Siebens-Drake Research Institute, London, Ontario, Canada N6G 2V4 Received 27 March 2006; returned to author for revision 27 April 2006; accepted 17 July 2006 Available online 7 September 2006

Abstract

The matrix (M) protein of vesicular stomatitis virus (VSV) plays significant roles in the replication of VSV through its involvement in the assembly of virus particles as well as by facilitating the evasion of innate host cell defense mechanisms. The presence of methionine at position 51 (M51) of the matrix (M) protein of the VSV Indiana serotype (VSVInd) has been proven to be crucial for cell rounding and inhibition of host cell gene expression. The M protein of VSVInd with the substitution of M51 with arginine (R:M51R) results in the loss of inhibitory effects on host cell gene expression. The VSVInd expressing the M(M51R) protein became the attractive oncolytic virus which is safer and more tumor-specific because the normal cells can clear the mutant VSVInd easily but tumor cells are susceptible to the virus because a variety of tumor cells lack innate antiviral activities. We have studied the role of the methionines at positions 48 and 51 of the M protein of the New Jersey serotype of VSV (VSVNJ) in the induction of cytopathic effects (CPE) and host cell gene expression. We have generated human embryonic kidney 293 cell lines inducibly expressing M proteins with M to R mutations at positions 48 and 51, either separately or together as a double mutant, and examined expression of heat shock protein 70 (HSP70) as an indicator of host cell gene expression. We have also generated recombinant VSVNJ encoding the mutant M proteins M(M48R) or M(M48R+M51R) for the first time and tested for the expression of HSP70 in infected cells. Our results demonstrated that the M51 of VSVNJ M proteins has a major role in cell rounding and in suppressing the host cell gene expression either when the M protein was expressed alone in inducible cell lines or when expressed together with other VSV proteins by the recombinant VSVNJ. Amino acid residue M48 may also have some role in cell rounding and in the inhibitory effects of VSVNJ M, which was demonstrated by the fact that the cell line expressing the double substitution mutant M(M48R+M51R) exhibited the least cytopathic effects and the least inhibitory effect on host cell gene expression. © 2006 Elsevier Inc. All rights reserved.

Keywords: Vesicular stomatitis virus; New Jersey serotype; Matrix protein; Methionin to arginine mutations; Cytopathic effects; Host gene expression;

Introduction indirect mechanisms by which the M protein induces CPE are disorganization of cytoskeletal elements such as actin or tubulin Vesicular stomatitis virus (VSV), the prototype virus of the by association with them (Blondel et al., 1990; Melki et al., 1994) Rhabdoviridae, induces typical cell rounding that leads to the and general inhibition of host cell gene expression (Black and lysis of infected eukaryotic cells (Simon et al., 1990). Although Lyles, 1992), which can result in the systemic breakdown of the the 48 nucleotide-long, plus-sense leader RNA or other cell by apoptosis (Kopecky and Lyles, 2003). components of VSV have been implicated as the cause of the VSV has two major serotypes: Indiana (VSVInd) and New CPE by VSV (Grinnell and Wagner, 1983; Kopecky et al., 2001), Jersey (VSVNJ). Matrix proteins of both VSVInd and VSVNJ the well documented cause of CPE has been the expression of the have 229 amino acids with about 59%–62% amino acid matrix (M) protein during VSV replication. The direct and sequence identity (Gill and Banerjee, 1986). In addition to the first methionine, 2 other methionines are conserved at positions ⁎ Corresponding author. Fax: +1 519 661 3359. 33 (M31) and 51 (M51) in both M proteins of VSVInd and E-mail address: [email protected] (C.Y. Kang). VSVNJ within the NH2-terminal 51 amino acids. It has been 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.07.022 42 G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 suggested that these two methionines may play a role in 2000; Stojdl et al., 2003). VSV expressing the M protein with producing truncated M proteins via internal translation initiation M51R mutation [M(M51R)] can be readily cleared from normal and in the induction of cell rounding by VSVInd (Jayakar and cells by an antiviral defense mechanism, whereas the trans- Whitt, 2002). The VSVNJ is further divided into two subtypes, formed cells which lack the antiviral activity are equally well Hazelhurst strain [VSVNJ(Haz)] and Ogden strain [VSVNJ infected and lysed by both the mutant and wild type VSV. These (Ogd)]. One additional methionine is present at position 48 in characteristics of the transformed cells and VSV expressing M the VSVNJ(Haz) M protein and 2 additional methionines are (M51R) have resulted in the mutant VSV being considered as a present at positions 21 and 48 in the VSVNJ(Ogd) M protein. safer and tumor-cell-specific oncolytic agent. The M48 is conserved in the M proteins of both VSVNJ(Haz) In this study, we examined the effects of the M33, M48 and and VSVNJ(Ogd) and is closely placed to the M51, indicating M51 of the VSVNJ M protein on host cell gene expression and that M48 may have a role in the cytopathogenesis induced by cell death. We generated tetracycline-inducible cell lines the M protein. expressing VSVNJ M mutants which contained individual It has been demonstrated that the VSV M protein inhibits the substitutions of arginines at positions M33, M48 and M51 and a expression of host cell proteins by inhibiting cellular transcrip- double substitution of both positions M48 and M51. Recombi- tion (Ahmed and Lyles, 1998; Black and Lyles, 1992) and by nant VSVNJ was recovered from cDNA for the first time, and blocking the nuclear export of a broad range of cellular RNAs the activity of mutant M proteins with the M48R or M48R+ including spliced mRNAs (Her et al., 1997). The amino acid M51R mutations encoded within the recombinant virus was residue in the M protein of VSVInd, which is critical for the also examined. Our results demonstrated that M51 of VSVNJ M inhibition of host cell gene expression, is the methionine at protein has a major role in the induction of cell rounding and position 51 (M51). The importance of the M51 for the inhibition inhibition of host cell gene expression either when the M of host cell gene expression was first indicated by the protein was expressed alone or when expressed together with conditional temperature-sensitive M mutant of VSVInd, tsO82 other VSV proteins. Amino acid residue M48 may be partially (Coulon et al., 1990). It has been demonstrated that M51 of responsible for the cell rounding and the inhibitory effect on VSVInd M protein has a major role in disrupting nucleocyto- gene expression, as demonstrated by the least cytopathic effects plasmic transport of mRNAs by binding directly to components and the least inhibitory effects on the host cell gene expression of the nuclear pore complex such as Nup98, one of the in the cell line expressing the double mutant M(M48R+M51R) nucleoporins (Enninga et al., 2002; Petersen et al., 2001, 2000; of VSVNJ. von Kobbe et al., 2000), and/or by interrupting the interaction between Nup98 and Rae1/mrnp41 (Faria et al., 2005), a Results shuttling mRNA export factor that is bound by M protein as well. Replacement of M51 with either arginine (R), alanine (A), Transient expression of wild type and mutant matrix proteins of leucine (L) or aspartic acid (D) reduced the inhibitory effect of VSVNJ M protein on the nuclear export of cellular RNAs (Petersen et al., 2000; von Kobbe et al., 2000). Matrix proteins of both VSVInd and VSVNJ consist of 229 VSV became an attractive oncolytic agent for cancer therapy amino acids with about 60% amino acid sequence identity. The because of its wide host cell range, rapid replication and mild NH2-terminal regions of M proteins from both serotypes are pathogenicity in humans. Because VSV M proteins having the highly basic, and the hydrophobic region and the PPPY motif M51R mutation lack the inhibitory effect on host cell gene that are involved in virus budding (Jayakar et al., 2000) are also expression, VSV expressing the mutant M protein loses the conserved. Both serotypes of VSV possess methionines at means to counteract the antiviral activity of host cells such as positions 33 and 51 within the NH2-terminal 51 amino acids of interferon (IFN) induction (Ahmed et al., 2003) and overall the M protein. The M33 and M51 of the VSVInd M protein have inhibition of translation mediated by PKR activation. The been shown to play important roles in cell rounding (Jayakar cancer cells are impaired with responsiveness to both type I and Whitt, 2002) and in inhibiting host cell gene expression (IFN-α,-β) and type II IFN (IFN-γ), which is one of the (Ahmed et al., 2003; von Kobbe et al., 2000). There is one important innate host defense mechanisms against viral additional methionine at position 48 in the VSVNJ M protein infection (Balachandran and Barber, 2004). In addition, (Fig. 1). In order to examine the importance of the methionines transformed cells have an increased level of eIF2Bε, a catalytic at positions 33, 48 and 51 in the CPE and inhibitory effects on subunit of eIF2B and a guanine nucleotide exchange factor the host cell gene expression by the VSVNJ M proteins, (Balachandran and Barber, 2004). The subunits of eIF2B are individual or combined methionine to arginine mutations were involved in converting the inactive form of eIF2·GDP to the introduced (Fig. 1). active eIF2·GTP, which is required to form a eukaryotic It has been demonstrated previously that the expression of translation initiation complex. The activity of eIF2B is normally VSVInd M proteins from eukaryotic expression vector is self- reduced in the non-tumor cells by the activation of IFN-induced limiting (Black and Lyles, 1992; Blondel et al., 1990) as it acts to RNA-dependent protein kinase PKR and eIF2α phosphoryla- inhibit its own transcription. Therefore, we cloned the wild type tion. The lack of innate defense mechanisms in transformed and mutant M gene cDNAs into two separate plasmid expression cells makes them more susceptible to the VSV infection than vectors in which the transcription is driven by the bacteriophage primary cells (Balachandran and Barber, 2004; Stojdl et al., T7 transcriptional promoter (pBKS-M) or by the eukaryotic G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 43

Fig. 1. Amino acid sequence comparison between M proteins of VSVInd and VSVNJ within 51 amino acids from the NH2-terminus and M to R mutations. Methionine residues are bold faced. Individual mutations at positions 33, 48 and 51 are shown as M33R, M48R and M51R and double mutation at positions 48 and 51 is shown as M48R+M51R.

CMV promoter (pcDNA5-M). T7 promoter mediated expression driven expression of M protein was only detectable in cells of M protein was examined by transfecting recombinant transfected with pcDNA5-M(M51R) and pcDNA5-M(M48R+ virus vTF7-3 infected BHK21 cells with cDNA plasmids of the M51R) (Fig. 2B). The results suggest that methionine at 51 of wild type and mutant M genes. CMV promoter mediated VSVNJ M protein has a critical role in inhibiting gene expression expression of M protein was examined by transfecting BHK21 by the host cellular RNA polymerase as has been demonstrated cells with pcDNA5-M clones without vTF7-3 infection. The by the M51 substitution of VSVInd M protein. transfected cells were labeled with 35S-met for 3 h at 20 h post transfection, and the M protein was immunoprecipitated using Replacement of methionine with arginine at positions 33, 48 polyclonal antiserum against VSVNJ whole virus. High levels of and 51 in the VSVNJ M protein does not affect the budding all mutant and wild type M proteins [M(wt)] were expressed by activity of VSVNJ M protein the T7 RNA polymerase that was provided by the recombinant vaccinia virus in the cytoplasm (Fig. 2A). Compared to the T7 It has been demonstrated that M to R mutations at positions promoter driven expression of M proteins, CMV promoter 33 and/or 51 in the VSVInd M protein do not affect the assembly

Fig. 2. Transient expression of VSVNJ wt and mutant M proteins. (A) Expression of M proteins mediated by the bacteriophage T7 promoter. BHK21 cells at 90% confluence in 6 well culture plates were infected at an MOI of 6 with recombinant vaccinia virus vTF7-3 expressing bacteriophage T7 DNA-dependent RNA polymerase. The infected cells were transfected with 7.5 μg of pBKS-MNJ(M33R), pBKS-MNJ(M48R), pBKS-MNJ(M51R) or pBKS-MNJ(M48R+M51R) and labeled with 30 μCi/ml 35S-met for 3 h at 20 h post transfection. The labeled cells were lysed with 400 μl of lysis buffer (10 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.4% Na- deoxycholate, 10 mM Na2EDTA), and M proteins were immunoprecipitated using polyclonal antiserum against VSVNJ. (B) Expression of M proteins mediated by the CMV promoter. BHK21 cells at 70% confluence in 100 mm culture dishes were transfected with 30 μg of pcDNA5-MNJ(wt), pcDNA5-MNJ(M33R), pcDNA5-MNJ 35 (M48R), pcDNA5-MNJ(M51R) or pcDNA5-MNJ(M48R+M51R) and labeled with 30 μCi/ml of S-met for 3 h at 20 h post transfection. The labeled cells were lysed, and M proteins were immunoprecipitated using polyclonal antiserum against VSVNJ. 44 G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53

33, 48 and 51 do not affect the assembly and budding function of the VSVNJ M protein.

VSVNJ M protein with M48R+M51R double mutation is the least cytopathic

Examination of the effects of the M protein on cell rounding and host cellular gene expression by transfection of DNA plasmid is not suitable because all cells are not transfected equally and the M protein can inhibit its own expression from the plasmid, resulting in overall insufficient effects on cells. In order to circumvent the self-limiting expression of M proteins from the plasmid DNA, other researchers have used mRNAs of the M protein gene that were prepared by in vitro transcription to transfect the cells (Ahmed and Lyles, 1998; Lyles and McKenzie, 1997). But the results from the transfection of mRNA can be variable depending on the transfection efficiency. In order to have all the cells in the culture dish express M protein and because a very small quantity of M protein can induce cytopathic effects (Black and Lyles, 1992), we generated tetracycline-inducible human 293 cell lines which express wild type M protein: M(wt), two single mutants: M(M48R) and M Fig. 3. Substitutions of methionines to arginines at positions 33, 48 and 51 in the (M51R), and one double mutant: M(M48R+M51R). The VSVNJ M protein do not affect its role in virus budding. VSVNJ panhandle type DI particle NJDI(227-188) was recovered by cotransfecting pTV-HDI(227-188), results shown in Fig. 2B suggest that M(wt) and M(M33R) pBKS-NNJ, pBKS-PNJ, pBKS-GNJ, pBKS-LNJ and pBKS-MNJ (wt or M to R have the same effect on host cell gene expression. Therefore, we mutants) as described previously (Kim and Kang, 2005). The presence of the DI were interested to know the outcome of M48 and M51 particle genomic RNA was detected by Northern blot analysis using the DI mutations to arginine. In order to examine the role of particle genome specific riboprobe. methionines at positions 48 and 51, separately and together, each inducible cell line was treated with 1 μg/ml of tetracycline for 24 h to induce M protein expression. The cell lysates were and budding of VSVInd (Black et al., 1993; Jayakar and Whitt, prepared to detect the expression of M proteins by Western blot 2002). In order to study the effects of M to R changes at analysis using polyclonal antiserum against whole VSVNJ. positions 33, 48 and 51 in the VSVNJ M protein on the assembly Most of the cells expressing M(wt) were rounded up and and budding of VSVNJ, we recovered VSVNJ panhandle DI detached (Fig. 4A-1), whereas cells expressing M(M48R) particles from the cDNA using the VSV reverse genetics system showed less cytopathic effect than the cells expressing M(wt) (Kim and Kang, 2005). Since the panhandle type VSV DI (Fig. 4A-3). Most of the cells expressing M(M51R) or M particle contains all the necessary cis-acting sequences at the 3′ (M48R+M51R) remained attached to the culture dish with only and 5′ termini of its genome for replication and budding, the slight cell rounding (Fig. 4A-5 and -7). Although the cell lines recovery of the DI particles using the wild type and mutant expressing M(wt) and M(M48R) proteins showed pronounced VSVNJ M proteins was ideal for this study. VSVNJ DI particles CPE indicating the presence of M proteins in the cells, we could containing bacteriophage λ sequences NJDI(227-188) (Kim and only detect the expression of M proteins in the cell lines Kang, 2005) were recovered from cDNA using either the wild expressing M(M51R) and M(M48R+M51R) by Western blot type or mutant VSVNJ M proteins as described in the Materials analysis (Fig. 4B, lanes 4 and 5). This result is consistent with and methods. The recovered DI particles were amplified once the previous observation that a minute quantity of M protein can by coinfecting BHK21 cells with an MOI of 3 of the standard be detrimental to host cells (Black and Lyles, 1992). Our result VSVNJ in a 6 well culture plate. At 15 h postinfection, 2 ml of demonstrates that M51 in the VSVNJ M protein plays a major culture fluid containing amplified DI particles and VSVNJ role in the inhibition of cell rounding in 293 cells whereas M48 standard virus was collected. Fresh BHK21 cells in a 6 well may have little effect on the cell rounding. culture plate were infected with 300 μl of the harvested culture It has previously been demonstrated that the M(wt) protein of media. At 5 h postinfection, the cells were lysed and DI particle VSVInd alone causes apoptosis in BHK cells and HeLa cells RNA was isolated as described previously (Kim and Kang, (Kopecky et al., 2001). The induction of apoptosis was closely 2005). The presence of bacteriophage λ sequences in the related to the presence of M51 in the M protein and inhibition of extracted DI particle RNA was determined by Northern blot host cellular gene expression by the M protein, which was analysis using a λ-specific riboprobe. Correct sizes of NJDI demonstrated using the M51R mutant. Therefore, we examined (227-188) genomic RNA were detected in all of the cells the effects on apoptosis of M to R mutations in the VSVNJ M infected with recovered DI particles (Fig. 3). This result protein at positions 48 and 51, both individually and together. demonstrates that the respective M to R mutations at positions Approximately 5×106 cells in a 60 mm culture dish were G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 45

Fig. 4. Mutations at positions M48 and M51 of VSVNJ M protein have the greatest impact on the reduction of cytopathic effects. Tetracycline-inducible human 293 cell lines expressing M(wt) or M proteins with M to R mutations at positions 48 and 51 individually and together were generated. The cell lines were treated with 1 μg/ml of tetracycline for 24 h to induce the expression of M proteins (A) Pictures were taken at 24 h after the treatment of cells with tetracycline at 125× magnification. (B) The expression of M proteins was examined by Western blot analysis using the cell lysate and polyclonal antiserum against VSVNJ. treated with 1 μg/ml of tetracycline for 24 h to induce M protein determine the caspase-3 activity by colorimetric assay (Fig. 5B). expression and cell lysates were used to isolate low molecular Both apoptotic assays demonstrated that M(wt) induces the weight DNAs for the DNA fragmentation assay (Fig. 5A) and to strongest apoptotic effect (Fig. 5A, lane 1 and Fig. 5B WTw/tet),

Fig. 5. VSVNJ M protein with double mutations at both M48 and M51 show the greatest reduction in apoptotic activity. (A) Low molecular weight DNA was isolated from the cell lines treated or untreated with tetracycline. The isolated DNA was resolved on a 1.5% agarose gel. (B) The caspase-3 activities in cell lines expressing M (wt) or mutant M proteins were examined as described in the Materials and methods. Caspase-3 activity is depicted as optical density (O.D.) unit which was read at the wave length 405 nm. The error bar represents the standard deviation of the mean from triplicate assays. The paired two tailed t test on samples of M(M51R) and M (M48R+M51R): P=0.0421. 46 G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 and the substitution of M48 with R reduced the apoptotic activity induction of the M protein. The M protein was immunopre- to about 2/3 level of the wild type M (Fig. 5A, lane 3 and Fig. cipitated using the antibody against VSVNJ. There was a slight 5B). The substitution of M51 with R either in the single mutant increase in the expression of HSP70 in the parental human M(M51R) or double mutant M(M48R+M51R) reduced the 293 cells after heat shock (Fig. 6, lanes 10 and 11). In the apoptotic activity of VSVNJ M close to background levels, and it absence of M protein expression, all cell lines expressed levels showed that the substitutions at both positions 48 and 51 led to a of HSP70 similar to those of the control parental 293 cells complete shutdown of caspase-3 induction (Fig. 5A, lane 5 and 7 (Fig. 6, lanes 2, 4, 6, 8, 10 and 11). The cell lines expressing and Fig. 5B). Therefore, we conclude that the methionines at M(M51R) and M(M48R+M51R) showed similar levels of positions 48 and 51 are required for efficient apoptosis of human HSP70 expression to those which were not treated with 293 cells expressing the VSVNJ M protein. tetracycline (Fig. 6, lanes 7 and 9). However, the expression of HSP70 was decreased significantly in cell lines expressing VSVNJ M protein with M48R+M51R mutation loses its M(wt) and M(M48R) (Fig. 6, lanes 3 and 5). inhibitory effect on host cellular gene expression Next we examined whether or not the M51R mutation continues to reduce cytopathogenicity and inhibition of host cell Human 293 cells express heat shock protein 70 (HSP70) gene expression in the presence of other VSVNJ proteins. In constitutively with or without heat treatment (Theodorakis and order to address this question, we generated full-length cDNA Morimoto, 1987) as shown in Fig. 6, lanes 10 and 11. Therefore, clones of VSVNJ and recovered the viruses containing M genes we chose the expression of HSP70 as an indicator to test the which encode M(wt), M(M48R) and M(M48R+M51R) inhibitory effect of wild type and mutant M proteins of VSVNJ separately. The cloning of the full-length genome (Fig. 7) and on host cell gene expression. recovery of VSVNJ from the cDNA clone were carried out by In order to examine the effects of the mutations of the M reverse genetics as described in Materials and methods. The protein on the expression of HSP70, the expression of M growth rates of recombinant VSVNJ, rWT, rM(M48R) and rM protein was induced first by treating the cell lines with 1 μg/ (M48R+M51R) were determined by infecting BHK21 cells and ml of tetracycline followed by heat shock at 44 °C for 10 min human 293 cells at an MOI of 3. Both wild type and mutant to induce the HSP70. The expression of HSP70 was viruses showed similar patterns of replication in both cell lines, determined by labeling the cell with 30 μCi/ml of 35S-met although the viruses replicated slower in the human 293 cells for 2 h at 3 h after heat shock and immunoprecipitating the compared to the BHK21 cells (Fig. 8). The VSVNJ expressing M HSP70 using a monoclonal antibody against human HSP70. (M48R) or M(M48R+M51R) replicated slower than the viruses The expression of M proteins in the same cell lines was expressing M(wt) during the first 6 h after infection (Fig. 8). The examined by labeling the cells with 35S-met at 20 h after the recombinant viruses reached a peak titer at 8 h postinfection when the viruses were grown in human 293 cells and maintained similar level of replication until 10 h postinfection (Fig. 8B). In order to examine the effect of VSVNJ expressing M (M48R) or M(M48R+M51R) on the host cellular protein synthesis, human 293 cells were infected with the recombinant VSVNJ at an MOI of 6 and the infected cells were labeled with 30 μCi/ml of 35S-met for 1 h at 8 h postinfection. Overall cellular protein synthesis and VSVNJ protein synthesis were examined by SDS-PAGE using equal amounts of cell lysates (15 μg), which was demonstrated by staining the gel with Coomassie blue (Fig. 9A) and by Western blot analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same samples (Fig. 9B, lower panel). The 35S-met labeled proteins were visualized by autoradiography (Fig. 9B, upper panel). The remaining cell lysates were divided into two aliquots for immunoprecipitation using the antibody against

Fig. 6. A methionine at position 51 of the VSVNJ M protein is critical for HSP70 and the antibody against VSVNJ. The substitution of M blocking host cell gene expression. (A) Human 293 cell lines expressing VSVNJ with R at positions 48 and 51 in the M protein made migration μ M(wt), M(M48R), M(M51R) and M(M48R+M51R) were treated with 1 g/ml of the M protein slightly faster than that of the wild type (Fig. 2), of tetracycline. After 4 h treatment with tetracycline, the cells were heat shocked for 10 min at 44 °C. The expression of HSP70 was determined by labeling the and the pattern was also shown in the cells infected with the cells with 30 μCi/ml of 35S-met for 2 h at 3 h after the heat shock and recombinant VSVNJ expressing M(M48R) and (M48R +M51R) immunoprecipitating the HSP70 using a monoclonal antibody against human (Figs. 9B and C, upper panel, lanes 3 and 4), demonstrating that HSP70. The expression of M protein in the same cell lines was determined after the recombinant viruses expressed mutant M proteins. The 35 the heat shock by labeling the cells with S-met at 20 h after the induction of M overall protein synthesis was maintained at about 60% and 25% protein. The M protein was immunoprecipitated using the antibody against of uninfected cells in the cells infected with rM(M48R +M51R) VSVNJ. HSP70 denotes heat shock protein 70. VSVNJ denotes the M protein expressed in the cells infected with wild type VSVNJ. M on the lower panel or rM(M48R) compared to the 2% to 12% protein synthesis in denotes the wild type and mutant VSVNJ M proteins. Tet denotes tetracycline. cells infected with wild type virus (Fig. 9B). The results G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 47

Fig. 7. Schematic representation of the generation of full-length cDNA clones of VSVNJ. The restriction enzyme sites in the vector (SapI and RsrII) and in the VSV genome (AvaI, PflMI and NdeI) for the assembly of the full length are shown on the top of the panel. The restriction enzyme sites that cut once (NotI and KpnI) and twice (PacI) were introduced into the untranslated regions at the P gene, M gene and G gene. The cDNAs of the mutant viruses rM(M48R) and rM(M48R+M51R) were generated by using the PacI and NotI sites to replace the wild type M gene. demonstrate that M(M48R+M51R) lost its inhibitory activity or with rM(M48R) (Fig. 9B, lane 4), which demonstrates that on the host cellular protein synthesis significantly and decreased inhibition of host cellular protein synthesis correlates substitution of M48R has slight effects on the host cell gene with increased VSV protein synthesis. The expression of expression in cells infected with VSVNJ. The overall VSV HSP70, an indicator for host cell gene expression, decreased to protein synthesis was also increased in cells infected with rM an undetectable or significantly low level in cells infected with (M48R+M51R) compared to the cells infected with wild type wild type VSVNJ and VSVNJ M(M48R) respectively (Fig. 9C,

Fig. 8. Single cycle growth curve of wild type and methionine to arginine substitution mutant viruses of VSVNJ. BHK21 cells (A) and human 293 cells (B) were infected at an MOI of 3 of the viruses. After 1 h of adsorption, the infected cells were washed twice and 500 μl of culture media was taken immediately from the culture dish as a sample at 0 h. The same volumes (500 μl) of culture media were taken and fresh media were added every 2 h until 10 h postinfection. Viral titer was determined by standard plaque assay. The viral titer at each time point represents the average of triplicate experiments. 48 G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53

Fig. 9. Recombinant VSVNJ expressing M(M48R+M51R) does not block cellular gene expression. (A) Human 293 cells were infected with wild type, recombinant wild type, rM(M48R) or rM(M48R+M51R) viruses at an MOI of 6, labeled with 30 μCi/ml of 35S-met for 1 h at 8 h postinfection. The cell lysates were analyzed by SDS- PAGE, and the gel was stained with Coomassie blue to demonstrate that equal quantities (15 μg) of samples were loaded. (B) The gel was dried and exposed to X-ray film. The amount of GAPDH in 15 μg of each cell lysate was determined by Western blot analysis using a polyclonal antibody against human GAPDH. The bar graph on the right side of the panel shows the percent expression of total protein synthesis compared to that of uninfected cells, which was determined by measuring the optical density of protein bands between large protein L and G of VSVon the autoradiographed film. The error bars on the graph represent the standard deviations of the mean from triplicate samples. (C) HSP70 and VSVNJ proteins were immunoprecipitated separately from the cell lysate using a monoclonal antibody against human HSP70 and polyclonal antibody against VSVNJ (Kim et al., 2002). The upper panel shows the VSVNJ proteins immunoprecipitated by mixed polyclonal antibodies against VSVNJ whole virus and antibody against VSVNJ L protein. The lower panel shows the expression of HSP70 in the same cells from which the VSVNJ proteins were detected. G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 49

lower panel, lanes 1, 2 and 3). The cells infected with VSVNJ rM the eukaryotic plasmid vector, or in tetracycline-inducible cell (M48R+51R) expressed levels of HSP70 equivalent to about lines, VSVNJ M proteins containing the M51R mutation, M 60% of uninfected cells (Fig. 9C, lanes 4 and 5). The results (M51R) and M(M48R+M51R), were detected in good quantity, demonstrate that the M51 in the VSVNJ M protein, as with the however, little to no M protein was detected in M(M48R) or M VSVInd M protein, plays a major role in the blocking of host cell (wt) expressing cell cells (Figs. 2B and 4B). This indicates that gene expression. The results also suggest that M48 may have a it is mostly the M51 of VSVNJ M protein which is involved in slight effect in blocking host cell gene expression. the inhibition of host cell gene expression as in VSVInd. M48 is conserved in M proteins of both VSVNJ(Haz) and VSVNJ(Ogd) Discussion strains. Although the single mutation at M48R does not affect the inhibitory effect of the VSVNJ M protein significantly, the VSV, a single stranded negative sense RNA virus, has substitution of both M48 and M51 together as in the M(M48R+ become a useful tool for the treatment of cancer (Balachandran M51R) caused an increased reduction of its inhibitory effects on et al., 2001; Ebert et al., 2005; Obuchi et al., 2003; Stojdl et al., host cell gene expression relative to that of the single mutation 2003) and for the expression of antigens in host animals to M51R (Figs. 4B and 6). This result demonstrates that both M48 induce immune responses (Egan et al., 2004; McKenna et al., and M51 may be involved in binding nuclear pore complexes 2003; Rose et al., 2001). As an oncolytic viral agent, VSV such as Nup 98 (von Kobbe et al., 2000). The apoptotic cell which encodes the mutant M protein that has lost its inhibitory death induced by the VSVInd M protein is closely related to the effect on host cell gene expression is gaining attention as an inhibition of host cell gene expression by the M protein alternative virus to wild type VSV because of its improved (Kopecky and Lyles, 2003; Kopecky et al., 2001). Therefore, safety (Ahmed et al., 2004; Ebert et al., 2004; Stojdl et al., the more the M protein loses its inhibitory effects on the host 2003). All of the works on VSV as an oncolytic virus and as a cell gene expression, the less it is capable of inducing CPE and foreign gene delivery vector have been performed using apoptosis, which was demonstrated by the M(M48R+M51R) VSVInd. Another serotype of VSV, VSVNJ, which is not of VSVNJ in our experiments (Figs. 4A and 5). Although the M neutralized by antibodies against VSVInd has not yet been protein with M48R mutation inhibits the production of HSP70 generated as a recombinant virus from cDNA. Here we have expression when it is expressed alone in the tetracycline- investigated the effects of M to R mutations at positions 33, 48 inducible cell line (Fig. 6), the cellular caspase-3 activity and and 51 of VSVNJ M protein on CPE and host cell gene subsequent DNA fragmentation was reduced significantly with expression using cell lines which express M protein, as well as the M48R mutation compared to the wild type M protein (Fig. recombinant VSVNJ which expresses the wild type M protein or 5). In addition, the M48R and M51R double mutation resulted the mutant M proteins. Double substitutions at M48 and M51 in a much higher level of M protein synthesis compared to the with R in the VSVNJ M protein, both in cells expressing the M51R single mutation in cell lines expressing the M proteins protein alone or from the recombinant VSVNJ, showed a drastic (Fig. 4B). These results indicate that not only the M51R reduction of inhibitory effects on host cell gene expression. mutation but also the M48R mutation is required for optimal The NH2-terminal region of the VSV M protein contains reduction of cytotoxicity in infected cells. Thus, it is desirable to important amino acid residues required to support the replica- change methionines at both positions M48 and M51 to arginines tion of VSV during virus particle assembly and escape from to generate VSVNJ as a gene expression vector or an oncolytic innate cellular antiviral activity. The PPPY motif from positions virus. 24 to 27 is critical for VSV budding from the cell surface Although VSV is a rapidly replicating virus which can infect (Craven et al., 1999; Jayakar et al., 2000). The translation tumor cells quickly, eventually humoral and cellular immune codons for M33 and M51 in the VSVInd M protein have been responses against VSV will be elicited in the animal host suggested as being used for the synthesis of two additional (Kalinke et al., 1996; Puddington et al., 1986; Yewdell et al., NH2-terminally truncated M proteins, which have been 1986). An animal infected with VSV develops immune suggested to be responsible for cell rounding (Jayakar and responses within 1 or 2 weeks (Kalinke et al., 1996) and starts Whitt, 2002). It has been demonstrated that the M51 of the to neutralize the VSV, which makes the cancer treatment or VSVInd M protein is involved in its association with foreign gene delivery via VSV incomplete. This hinders the nucleoporins, which leads to the blocking of nucleocytoplasmic efficacy of additional treatments for cancer therapy or boost transport of cellular RNAs and in inhibiting overall cellular immunizations for vaccination with the same vector. The two gene expression (Coulon et al., 1990; Petersen et al., 2000; von different serotypes of VSV, VSVInd and VSVNJ, show 50% Kobbe et al., 2000). The VSVNJ M protein has three amino acid identity in the glycoprotein (Gallione and Rose, methionines at the NH2-terminal region, M33, M48 and M51. 1983). Antibodies raised against one serotype of VSV do not M33 and M51 are conserved in both VSVInd M and VSVNJ M neutralize the other serotype of VSV (Cartwright and Brown, proteins. When the VSV M protein is expressed from a plasmid 1972). Therefore, others have used VSVInd as a vaccine vector containing a eukaryotic promoter, a very small quantity of M in which the glycoprotein was replaced with that of VSVNJ to protein can inhibit its own synthesis from the plasmid (Black minimize the problems arising from this immune response and Lyles, 1992; Blondel et al., 1990), which can reflect the against the viral vectors (Rose et al., 2001, 2000). Although the effect of inhibition of host cellular gene expression by the M VSVInd with G protein replaced with VSVNJ serotype is useful protein. When we expressed the mutant VSVNJ M proteins from in evading the humoral immune response, it will not prevent the 50 G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53

cellular immune response which can be triggered by nucleo- VSVNJ nucleocapsid (N), phosphoprotein (P) and Large or matrix proteins. The cellular immune responses protein (L) gene clones in the pBKS vector containing internal against VSV proteins other than the G protein may result in ribosome entry site (IRES) of the encephalomyocarditis virus incomplete immune responses against the antigen of interest or (EMCV) 5′-untranslated region (pBKS-IRES/NNJ,pBKS- failure to complete oncolytic treatment. Therefore, the genera- IRES/PNJ and pBKS-IRES/LNJ) were generated by inserting tion of additional recombinant VSV from another serotype can IRES sequences to the upstream of each gene. First, the IRES increase the efficacy of using VSVas an oncolytic virus and as a sequence was amplified from the pTM1 vector (Elroy-Stein et live viral vaccine vector. Our data show that VSVNJ encoding M al., 1989) by polymerase chain reaction (PCR) using the primers (M48R+M51R) loses its inhibitory effect on host cell gene IRES5′ and IRES3′ and then cloned into the pBKS vector using expression (Fig. 9), thereby making it a good candidate for more a NotI site, resulting in the pBKS-IRES. After confirmation of efficient cancer therapies. In addition, recombinant VSVNJ is an the correct IRES sequence in the pBKS-IRES, the IRES effective viral vector for the expression of foreign genes, which sequence was cut with NotI and inserted upstream of the VSVNJ can be used to minimize problems associated with preexisting gene cDNAs in the pBKS-NNJ, pBKS-PNJ and pBKS-LNJ, with immune responses against VSV itself. the interrupting sequences remaining between the IRES and the beginning of each gene. In order to use the 11th AUG sequence Materials and methods in the IRES as a start codon without interruption of any other sequence between the IRES and each gene sequence, the DNA Cells and viruses fragments containing continuous sequences from the end of the IRES to the beginning of the respective gene were amplified by BHK21 cells were cultured in Dulbecco's modified Eagle's megaprimer PCR (Sarkar and Sommer, 1990). The regions medium (DMEM) supplemented with 5% fetal bovine serum containing extra sequences were replaced by the megaprimer (FBS). Flp-In T-REx 293 cells (Invitrogen) were cultured in PCR products. Briefly, the megaprimers containing the 3′ 132 DMEM containing 10% FBS, 100 μg/ml zeocin and 15 μg/ml nucleotides (nts) of the IRES and beginning the NNJ,PNJ and blasticidine. Tetracycline-inducible Flp-In T-REx 293 cells LNJ gene sequences were amplified by the first PCR using a expressing VSVNJ wild type or VSVNJ M protein mutant were primer IRES-375 and pairing primers IRES-HN, IRES-HP and cultured in DMEM containing 10% FBS, 15 μg/ml blasticidine IRES-HL for respective genes. The first PCR products were and 400 μg/ml hygromycin B. The BHK cell line expressing T7 purified and used with primers HN BglII, HP MfeI and HL PflfI RNA polymerase, BSR T7/5 (Buchholz et al., 1999), was grown for the second PCR to amplify the continuous sequences in DMEM containing 5% FBS and 500 μg/ml geneticin. encompassing the 3′ IRES 132 nts and various lengths of N, P Recombinant VSVNJ was generated from the VSVNJ(Haz) and L gene 5′ sequences. strain (Dr. S. Emerson, NIH, Bethesda, MD). The recombinant The full-length cDNA clone of the VSVNJ genome was and wild type VSVNJ were purified 3 times by plaque picking constructed by assembling four RT-PCR products and three and amplified in BHK21 cells. Recombinant vaccinia virus PCR products into the transcription vector pTV (Kim et al., expressing T7 DNA-dependent RNA polymerase, vTF7-3 2002; Pattnaik et al., 1992). The 7 PCR products were put (Fuerst et al., 1986), was prepared in TK− 143 cells as together sequentially, and finally the NdeI digest from pBKS- described previously (Mackett et al., 1985). LNJ was cloned into the previously assembled cDNA clone (Fig. 7). The full-length cDNA clone of VSVNJ was named as pTV- Plasmids VSVNJ(rwt). The full-length antigenomic RNA of VSVNJ is encoded from the pTV-VSVNJ(rwt) by the T7 RNA polymerase. Construction of cDNA clones of VSVNJ(Haz) genes in the The restriction enzyme sites shown in Fig. 7 are included in pBluescript II KS vector (pBKS; Stratagen), pBKS-NNJ, pBKS- each fragment and were used to assemble the full-length clone. PNJ, pBKS-MNJ, pBKS-GNJ and pBKS-LNJ has been described Subclones of A, B, F and G fragments (Fig. 7) were generated in previously (Kim et al., 2002; Kim and Kang, 2005). Plasmids the pBKS vector by RT-PCR using the genomic RNA isolated encoding VSVNJ M protein mutants under the control of the T7 from the purified VSVNJ(Haz). The fragments of C, D and E promoter, pBKS-MNJ(M33R), pBKS-MNJ(M48R), pBKS-MNJ (Fig. 7) were generated by PCR using the cDNA clone of each (M51R) and pBKS-MNJ(M48R +M51R), were generated in gene as a template and were cloned into pBKS. The primers pBKS-MNJ by megaprimer PCR-based site-directed mutagen- (numbers 1 to 15) used for the RT-PCR and PCR are described esis (Sarkar and Sommer, 1990). The schematics and primers in Fig. 7. All subclones were sequenced and compared with the for the construction of mutant pBKS-MNJ are described in sequences of VSVNJ gene clones which were previously used supplement 1. Plasmids expressing VSVNJ wild type and successfully for the recovery of DI particles (Kim et al., 2002). mutant M under the control of the CMV promoter were Segment A was generated by the megaprimer PCR method constructed by inserting DNA fragments of M genes from the using three separate primers. A small, double-stranded DNA pBKS-MNJ wild type and the mutants into the NotI site of fragment containing the T7 transcriptional promoter and 3′ pcDNA5/FRT/TO (Invitrogen). The resulting plasmids were genomic terminal sequences was amplified by PCR using the designated as pcDNA5-MNJ(wt), pcDNA5-MNJ(M33R), pTV as a template and primers 1 and 2 (Fig. 7). The small PCR pcDNA5-MNJ(M48R), pcDNA5-MNJ(M51R) and pcDNA5- product was used as a megaprimer, pairing with primer 3 to MNJ(M48R +M51R). amplify the fragment A. G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 51

The cDNA clones of VSVNJ encoding M(M48R) and M until 95% confluency, at which time they were treated with (M48R+M51R), pTV-VSVNJ(M;M48R) and pTV-VSVNJ(M; 1 μg/ml of tetracycline for 24 h to induce the expression of M M48R+M51R), were generated by replacing the M(wt) gene proteins. Cell lysates were prepared to isolate low molecular with the M gene containing the respective mutations. The M weight DNA for the DNA fragmentation assay and to measure gene cDNAs containing the mutations were amplified using the caspase-3 activity in the cell lysate. For the DNA primers 8 and 9 (Fig. 7) and pBKS-MNJ(M48R) and pBKS-MNJ fragmentation assay, cells were lysed in 300 μl of TE/Triton (M48R+M51R) as templates. The full-length cDNA clone was buffer [0.2% (v/v) Triton X-100, 10 mM Tris pH 8.0 and 1 mM digested partially with PacI first and then completely digested EDTA]. The cell lysates were treated with RNase A (1.5 μlof with NotI. The PCR products were cut with the same restriction 10 mg/ml solution) for 1 h at 37 °C. Sodium dodecyl sulfate enzymes and cloned into the full-length cDNA clone. The (12.5 μl of 10% solution) and proteinase K (2 μlof20μg/ml nucleotide changes were confirmed by DNA sequencing. solution) were added to the cell lysates and were incubated for 1 h at 50 °C. The low molecular weight DNA was extracted by Generation of inducible cell lines expressing wild type or treating the lysate with the same volume of phenol/chloroform/ mutant VSVNJ M proteins isoamylalcohol and concentrated by precipitating the DNA with 0.1 volume of 3 M sodium acetate and 2.5 volume of 100% The human embryonic kidney 293 cells expressing M(wt) or ethanol. The DNA pellet was dissolved in dH2O. The DNA was M proteins with M to R mutations were generated by using the resolved on a 1.5% agarose gel containing ethidium bromide. Flp-In T-Rex Core Kit (Invitrogen) according to the manufac- Caspase-3 activities from the cell lysates were measured using turer's manual. Briefly, the Flp-In T-Rex 293 cell line the BD ApoAlert™ Caspase Colorimetric Assay kit (BD (Invitrogen) expressing the Tet repressor and containing the Biosciences Clontech) according to the manufacturer's proto- Flp recombinase target sequence in its genome was used as a col. Briefly, the cells were lysed in 100 μl lysis buffer, protein parental cell line. The 293 parental cell line was cotransfected concentration of the cell lysates was determined by the Bradford with the pcDNA5-MNJ(wt), pcDNA5-MNJ(M33R), pcDNA5- method using the Bio-Rad Protein Assay kit (Bio-Rad) and MNJ(M48R), pcDNA5-MNJ(M51R) or pcDNA5-MNJ(M48R+ 500 μg of each sample was measured for the caspase-3 activity. M51R) and pOG44 encoding Flp recombinase. Several cell All assays were done in triplicate. colonies which were formed in the presence of the selective agents blasticidine (15 μg/ml) and hygromycin B (400 μg/ml) Determination of protein expression were isolated, expanded and checked for the expression of M proteins in the presence of tetracycline (1 μg/ml). Expression of matrix proteins in inducible cell lines was determined by both Western blot analysis and immunoprecipi- Recovery of DI particles and recombinant VSVNJ from cDNA tation analysis. Western blot analysis was carried out using 10 μg of cell lysate and ECL Plus Western Blotting Detection VSVNJ DI particle NJDI(227-188) was recovered from reagents (Amersham Biosciences). The level of GAPDH in the 35 cDNA by cotransfecting pBKS-NNJ, pBKS-PNJ, pBKS-MNJ S-met labeled cell lysate was determined by Western blot (wt) or pBKS-MNJ(M-R mutants), pBKS-GNJ, pBKS-LNJ and analysis using a purified goat polyclonal antibody raised against pTV-NJDI(227-188) as described previously (Kim and Kang, a internal peptide of human GAPDH (Santa Cruz Biotechnol- 2005). Recombinant VSVNJ expressing wild type or mutant M ogy, Inc.). Determination of total cellular protein synthesis after protein was recovered from the plasmid DNA by the reverse viral infection, immunoprecipitation of M proteins, VSVNJ genetics method employing the BHK cell line expressing proteins and human heat shock protein 70 (HSP70) was carried bacteriophage T7 RNA polymerase BSR T7/5 (Harty et al., out after labeling the cells with 30 μCi/ml of 35S-met (1175 Ci/ 2001). BSR T7/5 cells were seeded in a 100 mm culture dish to mmol, PerkinElmer). VSVNJ proteins were immunoprecipitated be approximately 90% confluent in 24 h. The cells were with rabbit polyclonal antisera against VSVNJ total proteins and maintained in DMEM supplemented with 5% FBS without the L protein (Kim et al., 2002; Kim and Kang, 2005). HSP70 antibiotics. The transfection was carried out with Lipofecta- was immunoprecipitated with a mouse monoclonal antibody mine™ 2000 (Invitrogen) according to the manufacturer's against HSP70 (Santa Cruz Biotechnology, Inc.). protocol. Briefly, the BSR T7/5 cells were contransfected with 10 μg of pBKS-IRES/NNJ,10μg of pBKS-IRES/PNJ,5μgof Northern blot analysis pBKS-IRES/LNJ and 15 μg (1.6 pmol) of one of the following plasmids: pTV-VSVNJ(rwt), pTV-VSVNJ(M;M48R) or pTV- Northern blot analysis of DI particle genomic RNAs in the VSVNJ(M;M48R +M51R). The culture media was harvested infected cells was performed as described previously (Kim et when more than 50% of cells showed cytopathic effects, al., 2002). approximately 48 h after transfection. Acknowledgments Apoptosis assay We thank Rosanne Kang and Chad Michalski for correcting Inducible cell lines expressing M(wt), M(M48R), M(M51R) the manuscript. This study was supported by a grant from the and M(M48R+M51R) of VSVNJ were grown in 60-mm dish Curocom-Sumagen Company Limited. 52 G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53

References Gallione, C.J., Rose, J.K., 1983. Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular Ahmed, M., Lyles, D.S., 1998. Effect of vesicular stomatitis virus matrix protein stomatitis virus. J. Virol. 46 (1), 162–169. on transcription directed by host RNA polymerases I, II, and III. J. Virol. 72 Gill, D.S., Banerjee, A.K., 1986. Complete nucleotide sequence of the matrix (10), 8413–8419. protein mRNA of vesicular stomatitis virus (New Jersey serotype). Virology Ahmed, M., McKenzie, M.O., Puckett, S., Hojnacki, M., Poliquin, L., Lyles, 150 (1), 308–312. D.S., 2003. Ability of the matrix protein of vesicular stomatitis virus to Grinnell, B.W., Wagner, R.R., 1983. Comparative inhibition of cellular suppress beta interferon gene expression is genetically correlated with the transcription by vesicular stomatitis virus serotypes New Jersey and inhibition of host RNA and protein synthesis. J. Virol. 77 (8), 4646–4657. Indiana: role of each viral leader RNA. J. Virol. 48 (1), 88–101. Ahmed, M., Cramer, S.D., Lyles, D.S., 2004. Sensitivity of prostate tumors to Harty, R.N., Brown, M.E., Hayes, F.P., Wright, N.T., Schnell, M.J., 2001. wild type and M protein mutant vesicular stomatitis viruses. Virology 330 Vaccinia virus-free recovery of vesicular stomatitis virus. J. Mol. Microbiol. (1), 34–49. Biotechnol. 3 (4), 513–517. Balachandran, S., Barber, G.N., 2004. Defective translational control facilitates Her, L.S., Lund, E., Dahlberg, J.E., 1997. Inhibition of Ran guanosine vesicular stomatitis virus oncolysis. Cancer Cell 5 (1), 51–65. triphosphatase-dependent nuclear transport by the matrix protein of Balachandran, S., Porosnicu, M., Barber, G.N., 2001. Oncolytic activity of vesicular stomatitis virus. Science 276 (5320), 1845–1848. vesicular stomatitis virus is effective against tumors exhibiting aberrant p53, Jayakar, H.R., Whitt, M.A., 2002. Identification of two additional translation Ras, or myc function and involves the induction of apoptosis. J. Virol. 75 (7), products from the matrix (M) gene that contribute to vesicular stomatitis 3474–3479. virus cytopathology. J. Virol. 76 (16), 8011–8018. Black, B.L., Lyles, D.S., 1992. Vesicular stomatitis virus matrix protein inhibits Jayakar, H.R., Murti, K.G., Whitt, M.A., 2000. Mutations in the PPPY motif of host cell-directed transcription of target genes in vivo. J. Virol. 66 (7), vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a 4058–4064. late step in virion release. J. Virol. 74 (21), 9818–9827. Black, B.L., Rhodes, R.B., McKenzie, M., Lyles, D.S., 1993. The role of Kalinke, U., Bucher, E.M., Ernst, B., Oxenius, A., Roost, H.P., Geley, S., Kofler, vesicular stomatitis virus matrix protein in inhibition of host-directed gene R., Zinkernagel, R.M., Hengartner, H., 1996. The role of somatic mutation expression is genetically separable from its function in virus assembly. in the generation of the protective humoral immune response against J. Virol. 67 (8), 4814–4821. vesicular stomatitis virus. Immunity 5 (6), 639–652. Blondel, D., Harmison, G.G., Schubert, M., 1990. Role of matrix protein in Kim, G.N., Kang, C.Y., 2005. Utilization of homotypic and heterotypic proteins cytopathogenesis of vesicular stomatitis virus. J. Virol. 64 (4), 1716–1725. of vesicular stomatitis virus by defective interfering particle genomes for Buchholz, U.J., Finke, S., Conzelmann, K.K., 1999. Generation of bovine RNA replication and virion assembly: implications for the mechanism of respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential homologous viral interference. J. Virol. 79 (15), 9588–9596. for virus replication in tissue culture, and the human RSV leader region acts Kim, G.N., Choi, W.Y., Park, M., Kang, C.Y., 2002. Replication and as a functional BRSV genome promoter. J. Virol. 73 (1), 251–259. transcription of viral RNAs by recombinant L proteins of New Jersey Cartwright, B., Brown, F., 1972. Serological relationships between different serotype of vesicular stomatitis virus. Virus Res. 90 (1–2), 347–364. strains of vesicular stomatitis virus. J. Gen. Virol. 16 (3), 391–398. Kopecky, S.A., Lyles, D.S., 2003. The cell-rounding activity of the vesicular Coulon, P., Deutsch, V., Lafay, F., Martinet-Edelist, C., Wyers, F., Herman, stomatitis virus matrix protein is due to the induction of cell death. J. Virol. R.C., Flamand, A., 1990. Genetic evidence for multiple functions of the 77 (9), 5524–5528. matrix protein of vesicular stomatitis virus. J. Gen. Virol. 71 (Pt. 4), Kopecky, S.A., Willingham, M.C., Lyles, D.S., 2001. Matrix protein and 991–996. another viral component contribute to induction of apoptosis in cells infected Craven, R.C., Harty, R.N., Paragas, J., Palese, P., Wills, J.W., 1999. Late domain with vesicular stomatitis virus. J. Virol. 75 (24), 12169–12181. function identified in the vesicular stomatitis virus M protein by use of Lyles, D.S., McKenzie, M.O., 1997. Activity of vesicular stomatitis virus M rhabdovirus– chimeras. J. Virol. 73 (4), 3359–3365. protein mutants in cell rounding is correlated with the ability to inhibit host Ebert, O., Harbaran, S., Shinozaki, K., Woo, S.L., 2004. Systemic therapy of gene expression and is not correlated with virus assembly function. Virology experimental breast cancer metastases by mutant vesicular stomatitis virus in 229 (1), 77–89. immune-competent mice. Cancer Gene Ther. Mackett, M., Yilma, T., Rose, J.K., Moss, B., 1985. Vaccinia virus recombinants: Ebert, O., Harbaran, S., Shinozaki, K., Woo, S.L., 2005. Systemic therapy of expression of VSV genes and protective immunization of mice and cattle. experimental breast cancer metastases by mutant vesicular stomatitis virus in Science 227 (4685), 433–435. immune-competent mice. Cancer Gene Ther. 12 (4), 350–358. McKenna, P.M., McGettigan, J.P., Pomerantz, R.J., Dietzschold, B., Schnell, Egan, M.A., Chong, S.Y., Rose, N.F., Megati, S., Lopez, K.J., Schadeck, E.B., M.J., 2003. Recombinant rhabdoviruses as potential vaccines for HIV-1 Johnson, J.E., Masood, A., Piacente, P., Druilhet, R.E., Barras, P.W., and other diseases. Curr. HIV Res. 1 (2), 229–237. Hasselschwert, D.L., Reilly, P., Mishkin, E.M., Montefiori, D.C., Lewis, M.G., Melki, R., Gaudin, Y., Blondel, D., 1994. Interaction between tubulin and the Clarke, D.K., Hendry, R.M., Marx, P.A., Eldridge, J.H., Udem, S.A., Israel, Z.R., of vesicular stomatitis virus: possible implications in the Rose, J.K., 2004. Immunogenicity of attenuated vesicular stomatitis virus viral cytopathic effect. Virology 202 (1), 339–347. vectors expressing HIV type 1 and SIV Gag proteins: comparison of Obuchi, M., Fernandez, M., Barber, G.N., 2003. Development of recombinant intranasal and intramuscular vaccination routes. AIDS Res. Hum. vesicular stomatitis viruses that exploit defects in host defense to augment 20 (9), 989–1004. specific oncolytic activity. J. Virol. 77 (16), 8843–8856. Elroy-Stein, O., Fuerst, T.R., Moss, B., 1989. Cap-independent translation of Pattnaik, A.K., Ball, L.A., LeGrone, A.W., Wertz, G.W., 1992. Infectious mRNA conferred by encephalomyocarditis virus 5′ sequence improves the defective interfering particles of VSV from transcripts of a cDNA clone. Cell performance of the vaccinia virus/bacteriophage T7 hybrid expression 69 (6), 1011–1020. system. Proc. Natl. Acad. Sci. U.S.A. 86 (16), 6126–6130. Petersen, J.M., Her, L.S., Varvel, V., Lund, E., Dahlberg, J.E., 2000. The matrix Enninga, J., Levy, D.E., Blobel, G., Fontoura, B.M., 2002. Role of nucleoporin protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport induction in releasing an mRNA nuclear export block. Science 295 (5559), when it is in the nucleus and associated with nuclear pore complexes. Mol. 1523–1525. Cell. Biol. 20 (22), 8590–8601. Faria, P.A., Chakraborty, P., Levay, A., Barber, G.N., Ezelle, H.J., Enninga, J., Petersen, J.M., Her, L.S., Dahlberg, J.E., 2001. Multiple vesiculoviral matrix Arana, C., van Deursen, J., Fontoura, B.M., 2005. VSV disrupts the Rae1/ proteins inhibit both nuclear export and import. Proc. Natl. Acad. Sci. mrnp41 mRNA nuclear export pathway. Mol. Cell 17 (1), 93–102. U.S.A. 98 (15), 8590–8595. Fuerst, T.R., Niles, E.G., Studier, F.W., Moss, B., 1986. Eukaryotic transient- Puddington, L., Bevan, M.J., Rose, J.K., Lefrancois, L., 1986. N protein is the expression system based on recombinant vaccinia virus that synthesizes predominant antigen recognized by vesicular stomatitis virus-specific bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 83 (21), cytotoxic T cells. J. Virol. 60 (2), 708–717. 8122–8126. Rose, N.F., Roberts, A., Buonocore, L., Rose, J.K., 2000. Glycoprotein G.N. Kim, C.Y. Kang / Virology 357 (2007) 41–53 53

exchange vectors based on vesicular stomatitis virus allow effective Knowles, S., Marius, R., Reynard, J., Poliquin, L., Atkins, H., Brown, boosting and generation of neutralizing antibodies to a primary isolate of E.G., Durbin, R.K., Durbin, J.E., Hiscott, J., Bell, J.C., 2003. VSV human immunodeficiency virus type 1. J. Virol. 74 (23), 10903–10910. strains with defects in their ability to shutdown innate immunity are Rose, N.F., Marx, P.A., Luckay, A., Nixon, D.F., Moretto, W.J., Donahoe, S.M., potent systemic anti-cancer agents. Cancer Cell 4 (4), 263–275. Montefiori, D., Roberts, A., Buonocore, L., Rose, J.K., 2001. An effective Theodorakis, N.G., Morimoto, R.I., 1987. Posttranscriptional regulation of AIDS vaccine based on live attenuated vesicular stomatitis virus hsp70 expression in human cells: effects of heat shock, inhibition of protein recombinants. Cell 106 (5), 539–549. synthesis, and adenovirus infection on translation and mRNA stability. Mol. Sarkar, G., Sommer, S.S., 1990. The “megaprimer” method of site-directed Cell. Biol. 7 (12), 4357–4368. mutagenesis. BioTechniques 8 (4), 404–407. von Kobbe, C., van Deursen, J.M., Rodrigues, J.P., Sitterlin, D., Bachi, A., Wu, Simon, K.O., Whitaker-Dowling, P.A., Youngner, J.S., Widnell, C.C., 1990. X., Wilm, M., Carmo-Fonseca, M., Izaurralde, E., 2000. Vesicular stomatitis Sequential disassembly of the cytoskeleton in BHK21 cells infected with virus matrix protein inhibits host cell gene expression by targeting the vesicular stomatitis virus. Virology 177 (1), 289–297. nucleoporin Nup98. Mol. Cell 6 (5), 1243–1252. Stojdl, D.F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N., Yewdell, J.W., Bennink, J.R., Mackett, M., Lefrancois, L., Lyles, D.S., Moss, B., Bell, J.C., 2000. Exploiting tumor-specific defects in the interferon pathway 1986. Recognition of cloned vesicular stomatitis virus internal and external with a previously unknown oncolytic virus. Nat. Med. 6 (7), 821–825. gene products by cytotoxic T lymphocytes. J. Exp. Med. 163 (6), Stojdl, D.F., Lichty, B.D., tenOever, B.R., Paterson, J.M., Power, A.T., 1529–1538.