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Short Communication |
1 Centre for Gene Therapeutics, McMaster University, 1200 Main Street W MDCL-5023, Hamilton, ON L8N 3Z5, Canada
2 University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON, Canada
3 Ottawa Regional Cancer Centre, University of Ottawa, 503 Smyth Road, Ottawa, ON, Canada
Correspondence
Brian D. Lichty
lichtyb{at}mcmaster.ca
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ABSTRACT |
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; Clinton et al., 1978), as well as an important role in mediating budding of virions from the infected cell (Harty et al., 2001; Jayakar et al., 2000). The M protein also plays a crucial role in helping VSV to escape cellular antiviral programmes. This appears to be accomplished by blocking the expression of antiviral gene products (such as beta interferon) in response to infection, thereby allowing the virus to replicate unabated. This inhibition of cellular gene expression has been attributed to the ability of the M protein to block both cellular transcription (Black & Lyles, 1992) and nucleocytoplasmic transport (Petersen et al., 2000, 2001; von Kobbe et al., 2000). Indeed, it now appears that VSV employs this block in nucleocytoplasmic mRNA transport to thwart host innate immune mechanisms (Stojdl et al., 2003). This inhibition appears to involve an interaction between the M protein and the nuclear pore (von Kobbe et al., 2000) and is mediated via the mRNA export factor Rae1 (Faria et al., 2005). The cytopathic effect (CPE) induced by VSV infection has been attributed to the M protein and expression of M protein alone is capable of inducing CPE (Blondel et al., 1990; Kopecky & Lyles, 2003a, b; Kopecky et al., 2001). In most cells, this effect is due to inhibition of host-cell gene expression (Kopecky & Lyles, 2003b), whilst in others (e.g. L929 cells) M protein mutants allow expression of the death receptor apoptotic pathway by the infected cell (Gaddy & Lyles, 2005).
In an attempt to dissect the function of the N terminal portion of the M protein, we created a series of constructs fused to enhanced green fluorescent protein (EGFP) and expressed them in mammalian cells. In the course of this work, a fusion protein was created (in pEGFP-N1; Clontech) comprising the N-terminal 72 aa of the M protein fused to EGFP (WT+72–GFP) Surprisingly, WT+72–GFP showed perfect co-localization with mitochondrial markers, including anti-cytochrome c monoclonal antibody (Zymed) staining (Fig. 1a) and the mitochondrial marker OCT–DsRed2 (ornithine carbamyl transferase pre-sequence fused to DsRed2) (Harder et al., 2004) (Fig. 1b). To ensure that this localization was not an artefact of fusion to GFP, we constructed fusions with the unrelated fluorescent protein DsRed2 and once again observed targeting to mitochondria. Mitochondrial targeting was confirmed in U2OS, BHK and HeLa cell lines, as well as primary human foreskin fibroblasts (not shown).
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). Thus, the WT+91–GFP protein was targeted to the outer membrane or possibly the intermembrane space of the mitochondria.
When cells were transfected with full-length M protein fused to GFP, they displayed a diffuse green fluorescence in agreement with the reported distribution of M (Glodowski et al., 2002). This distribution fitted with the various functions attributed to this protein, but made it difficult to determine whether some of the M protein expressed in the cell was associated with the mitochondria. In order to specifically view full-length M51R M–GFP fusion protein associated with membrane-bound organelles such as the mitochondria, we employed a strategy for preferential permeabilization of the plasma membrane with digitonin. U2OS cells on coverslips were co-transfected with full-length M51R M–GFP and OCT–DsRed2 fusion constructs. As a negative control, cells were also co-transfected with EGFP and OCT–DsRed2. The next day, transfected cells were treated with digitonin (50 µg ml–1 in PBS) and plasma membrane permeabilization of cells was determined by staining briefly with trypan blue and viewing under a microscope. Coverslips were washed three times with ice-cold PBS, fixed and viewed. In cells co-transfected with M–GFP and OCT–DsRed2, most of the green fluorescence was removed from the cells by washing, whilst the red fluorescence was retained, indicating selective permeabilization of the plasma membrane. Under these conditions, much of the residual green fluorescence co-localized with the red mitochondrial marker, indicating that some of the full-length M–GFP fusion protein was in fact associated with mitochondria (Fig. 1d). Green fluorescence was lost entirely in all cases where control cells expressing EGFP and OCT–DsRed2 were treated in this manner (Fig. 1d).
Infected cells were subjected to fractionation by differential centrifugation using a standard protocol (Cavadini et al., 2002). The resulting nuclear, mitochondrial and cytoplasmic fractions were Western blotted and probed for M protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic marker (polyclonal antibody; Abcam), histone H3 as a nuclear marker (polyclonal antibody; Santa Cruz) and Cox IV as a mitochondrial marker (monoclonal antibody; Molecular Probes) (Fig. 1e). The results clearly demonstrated the presence of M protein in the washed mitochondrial pellet isolated from infected cells.
Further characterization of the mitochondrial-targeting signal demonstrated that the N-terminal fragments of M extending as far as aa 120 (of 229 aa) targeted fluorescent proteins to the mitochondria (Fig. 2a). In addition, fragments beginning at one of the alternative start methionines, M33 (M33–72–GFP), were also targeted to mitochondria, whilst fragments beginning at the other alternative start codon, M51 (M51–91–GFP), were not. However, a fragment including only aa 1–50 (WT+50–GFP) did not target GFP to mitochondria, indicating that the targeting motif spans M51, but does not require M51 (Fig. 2a), as also shown by deletion of M51 (WT+72 
M51–GFP). M33 was also not required, as demonstrated by the WT+72 M33A–GFP construct. It should also be noted that when these fragments were fused to the C terminus of GFP (GFP–WT+72 and GFP–WT+91), they failed to localize to the mitochondria. This is typical of mitochondrial-targeting motifs found at the N termini of proteins (Gordon et al., 2000).
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, sequences on both sides of M51 appeared to be required for targeting to the mitochondria. In order to define the targeting motif in more detail, we mutated clusters of amino acids in this region to alanine to attempt to define a mutation that failed to target the N terminus of the M protein to the mitochondria. The only mutations found to date that did so were 66FTV68
AAA, 66FT67AA and 66FA (see amino acid sequence in Fig. 2a). These mutations, in the context of the first 91 aa fused to GFP, led to a diffuse cellular distribution with some indication of targeting to the plasma membrane (Fig. 2c) compared with the punctate mitochondrial localization seen with the non-mutated WT+91–GFP construct (Fig. 2b). F66 is conserved across several strains of VSV and is present in a 
-sheet exposed on the surface of the crystal structure reported for VSV M protein (Gaudier et al., 2002).
It was noticed that when M–GFP or M–DsRed2 fusion proteins were expressed in cultured cells, many of these cells lost their usual tubuloreticular mitochondrial organization and displayed perinuclear punctiform mitochondria (Fig. 3a). In order to quantify the frequency at which this distribution was induced, cells were transfected with WT+91–GFP, WT33–91–GFP and FT-AA+91–GFP (containing the 66FT67AA mutation) expression constructs. Random microscopic fields were analysed and the proportion of cells displaying punctate or reticulotubular mitochondria was calculated for cells expressing these M–GFP fusion proteins or OCT–GFP alone as a control. At 24 h post-transfection, punctate mitochondria were evident in >60 % of cells expressing WT+91–GFP compared with 24 % of cells expressing WT33–91–GFP and 7 % of cells expressing the mutated FT-AA+91–GFP (Fig. 3b). The intermediate phenotype displayed by the WT33–91–GFP fusion protein may relate to an apparent reduced efficiency of mitochondrial targeting observed when the N-terminal 32 aa are absent (data not shown).
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). We also calculated the number of transfected cells displaying reduced MitoTracker staining. Forty-two per cent of cells expressing the WT+91–GFP fusion protein displayed reduced or undetectable MitoTracker staining and only 6 % of those cells retained a reticulotubular mitochondrial network. The other fusion proteins had minimal effects on MitoTracker staining (Fig. 3b). Reduced MitoTracker staining has been used previously as an indicator of reduced mitochondrial membrane potential (Jacotot et al., 2000). Thus, VSV M–GFP fusion proteins can alter the structure of mitochondria in transfected cells, leading to a loss of membrane potential.
To determine whether VSV infection induced similar alterations in mitochondrial organization, we transfected cells with the mitochondrial marker protein OCT–GFP and infected cells with a recombinant VSV expressing monomeric red fluorescent protein 1 (mRFP1, inserted into pXNDG as described previously; Stojdl et al., 2003) for identification of infected cells. Time-lapse images of live cells were collected. The mitochondria of infected cells were seen to undergo the same alterations in structure (Fig. 3e) as in cells co-transfected with WT+72–GFP and OCT–DsRed2 (Fig. 3d).
The M protein of VSV has several functions attributed to it and in this paper we have added the novel observation that this protein has a mitochondrial-targeting motif in its N terminus. Expression of fragments of M that localized exclusively to the mitochondria led to alterations in both structure and function of the mitochondria. Whilst only a small fraction of the full-length protein present in a cell can be localized to the mitochondria, it is intriguing to note that the mitochondria of infected cells undergo similar alterations.
Many viruses express proteins that localize, at least in part, to mitochondria and many of these proteins alter the apoptotic response of the infected cell (reviewed by Boya et al., 2004). In some cases, these proteins mediate mitochondrial effects very similar to those seen here with VSV M protein (Everett et al., 2000; McCormick et al., 2003). It is reasonable to predict that almost all viruses must evolve some means of dealing with the apoptotic response to infection induced in the host cell in order to carry out their normal life cycle. In fact, removal of this ability can lead to altered pathogenesis in vivo (Itoh et al., 1998; Mossman et al., 1996; Park et al., 2003). We are currently constructing mutant viruses with M protein mutations that prevent targeting of this protein to mitochondria to elucidate the role that this targeting plays in the life cycle of VSV.
Whilst VSV and indeed the M protein itself have been demonstrated to induce apoptosis (Kopecky & Lyles, 2003a, b), this effect is largely due to inhibition of host-cell gene expression (Kopecky & Lyles, 2003b). It is currently unclear whether the mitochondrial functions of the M protein contribute to this pro-apoptotic effect or are in fact working to inhibit this response. Whilst this virus has clearly evolved mechanisms to inhibit expression of host genes such as beta interferon in response to infection, it may have co-evolved a means of ‘managing’ the apoptotic response to this blockade in order to keep the muted host cell alive long enough to produce large numbers of progeny. Ultimately, VSV strains with mutations that prevent the M protein targeting the mitochondria will need to be generated to determine what role, if any, this cryptic mitochondrial-targeting motif has on the life cycle of VSV. To date, attempts described herein to isolate a virus bearing such mutations have failed, possibly indicating that mutation of the highly conserved phenylalanine at position 66 may yield a non-functional M protein. We are currently searching for mutations to less-well-conserved flanking amino acids that may allow the rescue of such a virus.
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REFERENCES |
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Received 12 December 2005;
accepted 26 June 2006.
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