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Matrix Protein of VSV New Jersey Serotype Containing Methionine To View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 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; Apoptosis 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).
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