In the present study, we examined specific amino acid changes associated with adaptation of CVB2O to cytolytic infection in RD cells. Our results showed that a single amino acid change on the capsid surface of CVB2O transforms the virus from a noncytolytic variant to a virus causing cytolysis. The characterization of the viral infection suggested that the CVB2O-induced cytolysis was associated with an apoptotic response (Fig. ).
FIG. 8. Schematic illustration of the CVB2O-induced apoptotic response in RD cells. Cytolytic CVB2O infection (vVP1Q164K) of RD cells results in activation of both the extrinsic (procaspase-8) and intrinsic (procaspase-9) pathways. These pathways are interconnected (more ...)
Previously published results have shown that CVB2O has the capacity to adapt to cytolytic replication in RD cells and that this novel property was associated with three nonsynonymous mutations (66
). The results from reverse genetics studies with the cloned single mutants presented here demonstrated that a single surface-exposed amino acid change (Q164K) in the VP1 capsid protein of this virus is sufficient for the transformation to a cytolytic phenotype. This observation supports the view that specific capsid residues influence picornaviral cell type specificity and tissue tropism (1
In detailed studies of the virus replication in RD cells by real-time PCR and titration on permissive GMK cells, the different cDNA clone-derived CVB2O variants showed similar properties regarding genome replication and virus progeny production. The productive infection of the cytolytic virus and noncytolytic CVB2O variants was further confirmed by immunofluorescence studies. This study also showed, together with the single-step growth curve and the observed onset of CPE, that there is a lag phase between the initial phase of viral replication and the CPE. The same phenomenon has previously been observed for another picornavirus, the Ljungan virus of the Parechovirus
). Possibly, this delay of CPE is a consequence of a virus that is not yet completely adapted to its host cell. However, the mechanism(s) involved in this delay of the CPE in RD cells infected with the cytolytic CVB2 variant remains to be elucidated. Taken together, the CVB2Owt and all CVB2O mutants were able to replicate in RD cells, a property that was not linked to cytolysis.
The nonstructural 2C protein of picornaviruses is a multifunctional protein with reported activities, including guidance of viral replication complexes to cytoplasmic membranes, enzymatic nucleotide triphosphatase activity, and involvement in virion assembly (48
). Results from analyses of the CVB2O mutant expressing a single 2C substitution (K185R) presented here suggested that this genetic change was not an essential determinant for the cytolytic phenotype of CVB2ORD. However, infection of RD cells with CVB2ORD, expressing all three adaptive substitutions, resulted in an earlier onset of cytolysis than infection with vVP1Q164K
. Thus, the additional substitution in VP1 (I118F) together with the amino acid change of 2C contributed to the cytolytic phenotype in RD cells by a mechanism that remains to be elucidated.
Previously, it has been shown that CVB can establish persistent infections in RD cells (4
). Monitoring of repeated passages of the wild-type CVB2O in RD cells revealed a continuous release of infectious progeny although no signs of CPE were observed. The ability of the CVB2O wild-type strain to replicate in RD cells without evident signs of CPE has, to our knowledge, never been characterized before. Studies of poliovirus have shown that persistent infections may be established when HEp-2 cells are subjected to virus at a very low MOI (62
). In contrast, the CVB2O infection of RD cells seems to be independent of virus dosage since these cells remained persistently infected even when they were exposed to a very high viral dose (MOI of 100). The release of noncytolytic CVB2Owt is possibly facilitated by viral proteins such as nonstructural protein 2B, which has been shown to modify membrane permeability (23
). Conclusively, these results suggest that CVB2O established a persistent infection in cultured RD cells. This may have implications for CVB2 infections in vivo
where muscle cells possibly serve as virus reservoirs. Indeed, CVB2 RNA has been detected in muscle tissue of patients with chronic muscle diseases (3
The virus-host cell system represented by the different CVB2O variants and the RD cells provided a well-defined model system for studies of persistent and cytolytic CVB2O infection. This model system also made it possible to examine whether apoptosis played a role during CVB2O infection in these cells. Theiler's murine encephalomyelitis virus is highly cytolytic in permissive BHK-21 cells, causing rapid cell destruction without signs of apoptosis; however, in less permissive cells, virus growth is markedly reduced, and viral replication is accompanied by induction of apoptosis (40
). Other picornaviruses, including CVB3, enterovirus 71, foot-and-mouth disease virus, poliovirus and avian encephalomyelitis virus, have also been shown to interact with the cellular apoptotic pathway (17
). Although one-step growth analysis demonstrated that RD cells were equally susceptible to replication of the cytolytic and persistent CVB2O variants, major disparities were revealed when the apoptotic status of infected cells was examined. Prior to cell cytolysis, distinctive apoptotic hallmarks, i.e., extensive DNA degradation and activation of caspase-8, caspase-9, and caspase-3, were observed in RD cells infected with the cytolytic CVB2O (vVP1Q164K
) variant. In addition, the infection was accompanied by an activation of Bid, an activation previously described for RD cells infected with enterovirus 71 (19
). Conversely, RD cells persistently infected with the parental CVB2O virus showed no signs of DNA degradation and caspase activation. In conclusion, these data add to the increasing knowledge of the interplay between picornaviruses and the cellular apoptotic pathway during infection.
Several reports have suggested that the structural proteins of picornaviruses are involved in the induction of apoptosis (33
). For example, the VP1 protein of foot-and-mouth disease virus has been shown to activate a proapototic response by binding to integrins and by deactivation of the Akt signaling pathway (63
). In other experiments, the VP3 protein of avian encephalomyelitis virus colocalized with mitochondria (50
), whereas the VP2 protein of CVB3 interacted with a proapoptotic factor, called Siva (33
). Hence, picornaviral structural proteins are associated with induction of apoptosis by different mechanisms. Whether any of these mechanisms are involved in the apoptotic response associated with the VP1 protein of CVB2O remains to be elucidated.
Although induction of apoptosis in RD cells was linked to caspase activation, the involvement of other apoptotic mechanisms cannot be excluded. For example, the large quantities of viral products that accompany a poliovirus infection result in drastic rearrangement of the endoplasmic reticulum (ER) (72
). ER-mediated stress resulting in activation of apoptosis has previously been reported for cells infected with a virus of the Flaviviridae
family, the Japanese encephalitis virus (77
). In addition, a previous study has shown that caspase inhibition does not prevent cell death of CVB3-infected HeLa cells (17
). Thus, apoptotic activity is certainly not the only mechanism that can induce cell death and cytolysis during picornavirus infections. For example, the multifunctional 2A and 3C proteins are involved in the breakdown process of the cytoskeleton at the time of cell lysis (5
). Consequently, cell lysis induced by a virus infection is probably a result of several different intracellular processes.
CVB infections cause myocarditis and pancreatitis (31
). As suggested by serological studies, these viruses also appear to be associated with insulin-dependent diabetes mellitus (38
). Several experimental murine models have been developed to study CVB infections. In this report, a comparative study in a mouse model indicated that the cytolytic CVB2ORD variant induced inflammation in the exocrine pancreas while no signs of inflammation were observed in pancreatic tissue of mice infected with the parental CVB2O strain. This pancreatic virulence is consistent with other studies of CVB infections in mice, which have shown that virus-induced inflammation was located to the exocrine pancreas and not the endocrine tissue (70
). However, these initial observations, including the possible association between the cytolytic RD phenotype and the increased propensity to induce pancreatic inflammation in mice, need to be explored further.
Knowledge about virus-host cell interactions is important in order to elucidate mechanisms by which the virus causes damage and to enable development of new antiviral treatments. The present study illustrates the adaptive potential of picornaviruses, where a single capsid substitution in CVB2O played a pivotal role for both cytolytic infection and induction of an apoptotic response in RD cells. In the absence of this amino acid change, CVB2O established a persistent, noncytolytic infection without signs of apoptosis. Thus, this study shows that the fate of the infected cell depends on a complex balance between the host and the virus and that this balance can be disrupted by a single substitution.