Apoptosis is inversely correlated with the pathogenicity of lyssaviruses (32
). Studies of neuronal cell cultures show that some lyssavirus genotypes, in particular, LAG and MOK, induce cell death, unlike the classical rabies virus of genotype 1 (THA), which does not kill host cells (15
). The lyssavirus glycoprotein has an important role in the induction of apoptosis (10
). However, the viral M protein is also able to induce apoptosis in a manner independent of that for other viral proteins via a TRAIL and caspase 8-dependent pathway (22
). A comparison of M proteins from different lyssaviruses indicated that the M protein from MOK, a genotype 3 virus with low virulence, induces cell death by dysregulating the respiratory chain (15
). In cells expressing M-MOK, CcO activity was significantly inhibited and caspase 9 was activated. A segment of M-MOK (aa 46 to 110) also retains the CcO-inhibiting and apoptosis-inducing activities through the TRAIL-dependent mechanism of the intact molecule (15
). The aim of the present study was to describe the apoptotic functions of M-MOK in more detail and to identify the domains of the protein involved in these functions. Using a strategy of deletion and site-directed mutagenesis, we identified a domain of 20 residues (aa 67 to 86) in M-MOK that retained the capacity to induce TRAIL-mediated cell death and inhibit CcO activity.
Our mutation analyses show that the K at position 77 in M-MOK is essential for inhibition of CcO activity: M-THA mutants presenting K77 inhibited CcO activity and all the mutations of K77 except a K-to-R conserved-charge mutation in a small subfragment spanning aa 67 to 79 restored the CcO activity. Similarly, the N at position 81 in M-MOK is necessary to induce apoptosis via the TRAIL signaling pathway. Supernatants of cells transfected with constructs expressing apoptotic activity induced TRAIL-mediated cell death in nontransfected cell cultures; the factor responsible was therefore presumably soluble and secreted into the supernatant.
TRAIL is a type II membrane cytokine with a releasable extracellular component that is capable of receptor recognition and apoptosis induction (24
). Although death receptors are constitutively expressed in most cell types, expression of their ligands is tightly regulated. Several viruses cause sensitivity to TRAIL or its expression and release from infected cells (6
). Sensitivity to TRAIL is influenced by regulation of TRAIL receptor expression by the p53 tumor suppressor protein (16
). The induction of type I interferon by viral infection increases the level of expression of TRAIL in various cell types (11
), and this can result in cell death from infiltrating immune effector cells (4
). However, the mechanism involved during lyssavirus infection does not seem to be related to the overexpression of TRAIL (22
; unpublished results). We report on the sensitization of cells to TRAIL after expression of the M1-7 construct, resulting in cell suicide and fratricide. Neutralizing antibodies prevented cell death in this system, indicating that the increased apoptosis is most probably related to a higher level of transduction of the signal generated by constitutive TRAIL. It would be of interest to investigate whether the low TRAIL sensitivity of THA-infected cells is also due to antiapoptotic factors, such as cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (cFLIP) (31
Functional signal motifs within pluripotent matrix proteins can be overlapping or situated in close proximity to each other. The active residues at positions 77 and 81 in M-MOK participate in both induction of TRAIL-mediated apoptosis and inhibition of CcO activity. These residues are located at the surface (Fig. ) of the recently resolved crystal structure of a lyssavirus M protein and are spatially independent of other known functional motifs (2
). The effects of these residues on apoptosis and CcO activity could be due to adjacent overlapping motifs or to interaction with points of cross talk between the two signaling pathways, such as B-cell lymphoma 2 (Bcl-2) family members or inhibitor of apoptosis (IAP) proteins (1
). The signaling pathways are not always mutually exclusive, and several death receptors activate both proapoptotic and survival pathways.
FIG. 5. Representation of the structure of the LAG matrix protein from residues 48 to 202 after a crystallography study (2). Positions K77 and N81 are at the outside face of the protein.
Some viral disease mechanisms involve apoptosis in the CNS (33
). However, the rabies virus avoids early apoptosis (21
). Mutation analyses of M-MOK and M-THA indicate that mutations at positions 77 or 81 modify the activity of the molecule (Fig. ). However, exchanging the amino acids at positions 77 and 81 of M-THA with those present in M-MOK did not completely confer the apoptotic activity of M-MOK on M-THA. Therefore, other as yet unidentified motifs within the protein are presumably involved.
Alignments of the M-protein sequences from the seven described lyssavirus genotypes showed that most strains of genotype 1 have R and E at positions 77 and 81, respectively; strains of genotypes 2 and 3 have K and N at these respective positions (Fig. ) (3
). Therefore, residues R77 and E81 in genotype 1 may correlate with delayed apoptosis and, subsequently, with higher pathogenicity. Indeed, most isolates from genotypes 2 and 3 have lower pathogenicity (30
) than isolates from genotype 1 (3
). These correlations are consistent with the M protein being important in the modulation of the host response to infection and especially the delayed innate immune response after infection with genotype 1 virus. This delay may be essential for viral infection and colonization of the CNS. The cell death induction process described here would benefit the host by limiting viral dissemination. The sequential induction of TRAIL-mediated apoptosis, particularly the delayed induction observed in the case of THA, is intriguing (15
). It remains to be explored whether this delay favors dissemination of THA into the central nervous system, leading to a pathogenesis more severe than that of MOK. This is striking when it is considered that THA is a highly pathogenic dog rabies virus isolated in Asia, where there is a high incidence of rabies, and that MOK, a wild isolate from Africa, is less pathogenic in humans (only two cases in humans have so far been described in the literature) (8