This work was aimed primarily at studying the relationship between the two death programs, apoptosis and CPE, triggered by poliovirus infection of HeLa cells. Even minimal amounts of the viral proteins produced by translation of the input viral RNA were sufficient to turn on the apoptotic program, as evidenced by the development of apoptosis in the cells infected in the presence of guanidine. The proportion of infected cells committed to apoptosis increased roughly linearly during the first 1.5 to 2 h p.i., provided further expression of the viral genome was severely hampered. However, events occurring during the next stage of viral reproduction (after 2 h p.i.) changed the destination of the infected cells toward CPE. Thus, a major observation of this study was the occurrence of a switch in the commitment of the poliovirus-infected cells in the middle of the infectious cycle (Fig. ). Obviously, the outcome of competition between the two death programs triggered by infection depended nonlinearly on the expression of the viral genome.
Schematic representation of the time course of commitment of poliovirus-infected HeLa cells to apoptosis and CPE. The type of death and the proportion of dying cells depend on the time of addition of inhibitors of viral gene expression.
Another regulatory switch occurring in the middle of infection is the development of an antiapoptotic state. Indeed, cells undergoing productive poliovirus infection could be demonstrated, before the appearance of CPE, to be resistant to nonviral apoptotic stimuli (34
). Also, as shown here, activation of caspase(s) is suppressed in the course of productive infection.
The early commitment of infected cells to apoptosis may be regarded as a defensive measure aiming at elimination of such cells prior to completion of the viral reproductive cycle. In other words, the cell may posses a very sensitive sensor(s) able to recognize limited amounts of a virus-specific product(s) and to turn on the suicide program. On the other hand, poliovirus has evolved a counterdefensive mechanism activating an antiapoptotic program.
Obviously, the complex pattern of alterations in the infected cells depends on the interplay between virus-specific and host components. With regards to the viral products, the switch from the proapoptotic state to the antiapoptotic state may involve several not mutually exclusive mechanisms. It is not unlikely that the completely processed, “mature” polypeptides may be underrepresented at early steps, and one may speculate that the effects of “immature” and “mature” viral proteins on the apoptotic system as well as on CPE development may be different. On the other hand, it may be assumed that a lesser amount of viral proteins is required to turn on the apoptotic program than to trigger development of the antiapoptotic state and to commit cells to CPE. Also, the expression of proapoptotic and antiapoptotic poliovirus functions may be differently related to the rearrangement of intracellular infrastructure known to develop in the middle of the infectious cycle (22
The commitment to apoptosis is accompanied by a relatively early (e.g., by 3 h p.i.) activation of a DEVD-specific caspase(s). This group of caspases includes caspase 3, which is believed to be an enzyme involved primarily in the final execution step of apoptosis (28
). It is likely that caspase 3 also plays this role in poliovirus-induced apoptosis, as it does in the apoptosis induced by some other RNA-containing viruses (6
). However, the involvement of another caspase with a similar substrate specificity (e.g., caspase 7) cannot be excluded either. The data available do not permit the discrimination between two possible causes of a low-level activation of a DEVD-specific caspase(s) in productively infected cells. It may reflect either (i) implementation of the apoptosis execution step in a small proportion of truly apoptotic cells present in any (even uninfected and nontreated) population of HeLa cells, or (ii) “footprints” of the interrupted commitment of the majority of the cells to apoptosis that developed early in infection.
Since no appreciable amount of a free caspase inhibitor could be detected in the productively infected cells, it seems reasonable to assume that viral products accumulating after 2 h of infection (or appropriate virus-induced cellular rearrangements) prevented activation rather than directly inactivated the enzyme. As a result, the implementation of the apoptotic program is interrupted. The research aimed at identification of the viral antiapoptotic protein(s) as well as at elucidation of the mechanism(s) involved is under way.
An important point concerns relationships between the induction of the antiapoptotic state and commitment to CPE. Although at the moment we are aware of no means to uncouple these two processes in the virus-infected cells, the antiapoptotic activity of certain virus-specific proteins in uninfected cells (N. Neznanov et al., unpublished data) suggests that they reflect distinct viral functions.
In the system described, the type of death of the poliovirus-infected cells, apoptosis or CPE, was related to the abortive or productive character of infection, respectively. However, this should not necessarily be the case in other poliovirus-cell systems. Since the apoptotic and CPE programs depend nonlinearly on expression of the viral genome, quantitative differences in the rates of viral translation and/or replication may result in qualitative changes of the infection outcome. The real situation is even more complicated, and in fact hardly predictable, due to intervention of a multitude of host proapoptotic and antiapoptotic factors, as evidenced for example by experiments with the Bcl-2-expressing cells. Our preliminary observations suggest that in certain host cells productive poliovirus infection might be accompanied by apoptosis, whereas in other host cells abortive infection failed to trigger apoptosis (unpublished data). Thus, the model shown in Fig. reflects the situation in certain cells but will not necessarily be as “clean” in others. Also, there is a claim that, in the central nervous systems of mice undergoing poliovirus-induced paralytic disease, infected neurons may die of apoptosis (15
). A system in which some apoptotic manifestations resulted from productive infection with another enterovirus, coxsackievirus B3, has recently been reported (9
), but again, this is not necessarily the case in other coxsackievirus systems. Nonuniform susceptibility of different host cells to the apoptosis-inducing activity of a given virus is perhaps a general phenomenon (see references 12
, and 27
). Furthermore, more or less related viruses may differ in their proapoptotic and/or antiapoptotic activities (7
), and some viruses (e.g., certain strains of Theiler's murine encephalomyelitis virus) may express unique antiapoptotic proteins (14
Such a complex control of the fate of infected cells should obviously have important implications for pathogenesis of viral diseases (reviewed in reference 16
). For example, the ability of picornaviruses (15
) and some other neuropathogenic RNA-containing viruses (e.g., 19
) to induce apoptosis in the central nervous system should likely be of clinical importance. Indeed the pattern of the disease should be affected, in particular, by the strength of the inflammatory reaction, which in turn should vary depending on the type of death of the infected neural cells (apoptotic cells do not generally elicit an inflammatory response).