In this study, we describe the identification of a novel coiled-coil 60-kDa protein, Beclin, which interacts with the cell death regulator Bcl-2. We demonstrate that Beclin overexpression in virally infected neurons in vivo results in substantial protection against Sindbis virus-induced disease. Mice infected with recombinant Sindbis viruses that express wild-type Beclin have less neural cell apoptosis, decreased viral replication, and a significant lower mortality rate than mice infected with recombinant viruses that express Beclin deletion mutants. Furthermore, the Bcl-2-binding domain of Beclin is required for the antiviral, antiapoptotic, and survival-promoting effects of Beclin on CNS Sindbis virus infection. These findings suggest that Beclin, via interactions with Bcl-2-like molecules, can function in vivo in the CNS as an antiviral host defense molecule.
Our conclusion that the protective, antiviral effects of Beclin are mediated through interactions with Bcl-2 (or Bcl-2-like molecules that bind to same region of Beclin as Bcl-2) is supported by data obtained with a chimeric Sindbis virus expressing Beclin lacking aa 88 to 151. Amino acids 88 to 150 of Beclin are sufficient to mediate an interaction with Bcl-2 and Bcl-xL in the yeast two-hybrid assay, and deletion of these amino acids from full-length Beclin significantly decreases the strength of the Beclin-Bcl-2 interaction in mammalian cells. The Sindbis virus construct expressing Beclin lacking aa 88 to 150 (SIN/beclinΔBcl-2BD) replicated to as high levels in mouse brain, resulted in as much apoptotic neural cell death, and resulted in the same mortality in mice as a control recombinant virus which expressed a 90-aa truncated Beclin protein (SIN/beclinstop). The lack of protective activity of BeclinΔBcl-2BD compared to full-length Beclin cannot be attributed to lower levels of BeclinΔBcl-2BD expression after infection with SIN/beclinΔBcl-2BD. The levels of flag-BeclinΔBcl-2BD were equal to or greater than levels of flag-Beclin detected by immunoblot analysis of lysates from BHK cells infected with SIN/beclin and SIN/beclinΔBcl-2BD, respectively. In addition, SIN/beclinΔBcl-2BD-infected mouse brains had more flag-immunoreactive cells than mouse brains infected with SIN/beclin. Thus, the lack of protective activity of BeclinΔBcl-2 is most consistent with the hypothesis that the antiviral effects of Beclin are mediated through interactions with Bcl-2-like proteins.
Some of the data in the present study suggest that the virus SIN/beclinΔBcl-2BD may be more neurovirulent than the virus SIN/beclinstop. Mice infected with SIN/beclinΔBcl-2BD had more virus RNA-positive cells in their brains at days 2 and 4 after infection than mice infected with SIN/beclinstop. Corresponding to the more rapid spread of infection, more cell death was seen earlier on after infection in the brains of mice infected with SIN/beclinΔBcl-2BD compared to SIN/beclinstop. Although the overall mortality rates of mice infected with SIN/beclinΔBcl-2BD versus SIN/beclin stop did not differ, but the mean day of death was slightly lower in mice infected with SIN/beclinΔBcl-2BD versus SIN/beclinstop. These observations raise the possibility that Beclin lacking the Bcl-2-binding domain functions as a dominant negative mutant. As a corollary, the naturally occurring Beclin isoform found in brain that lacks the Bcl-2-binding domain may serve as a death-promoting molecule.
Beclin may exert antiviral effects via interactions with either Bcl-2, Bcl-xL
, or yet unidentified Bcl-2 family members. In yeast two-hybrid assays, we found that the same region of Beclin interacted with Bcl-2 and Bcl-xL
and that loss-of-function mutations in the conserved BH1 domains of both Bcl-2 and Bcl-xL
blocked binding to Beclin. While both Bcl-2 and Bcl-xL
are important regulators of apoptosis in neurons (reviewed in reference 23
), we have found that Bcl-2, but not Bcl-xL
, protects mice against fatal encephalitis and delays Sindbis virus-induced death of cultured rat dorsal root ganglion neurons (14
). Thus, Bcl-2 may be more important than Bcl-xL
as a regulator of Sindbis virus-induced death in neural cells. Additional studies examining the effects of Beclin on Sindbis virus infection in mice that are deficient in Bcl-2 may help define whether Bcl-2 is the biologically important Beclin-binding partner in in vivo antiviral pathways in the CNS.
The mechanisms by which Beclin, in cooperation with Bcl-2-like proteins, functions to inhibit Sindbis virus replication and Sindbis virus-induced neuronal death are unknown. Both Semliki Forest virus and Sindbis virus have been shown to inactivate the antiapoptotic function of Bcl-2 in transfected fibroblasts through caspase-induced Bcl-2 cleavage (10
). In addition, Nava et al. have shown that crmA
increases the survival of Sindbis virus-infected mice (24
), suggesting a role for crmA
-inhibitable CNS caspase activity in the pathogenesis of fatal encephalitis. One possibility, therefore, is that binding to Beclin somehow protects Bcl-2 (or Bcl-xL
) from cleavage by caspases, thereby preventing Sindbis virus-induced cell death. A second possibility is that the Beclin–Bcl-2-like protein complex blocks an endoplasmic reticulum stress signal triggered by the Sindbis virus E2 and E1 envelope glycoproteins. One of the mechanisms by which Bcl-2 and Bcl-may inhibit apoptosis is by regulating the permeability of intracellular membranes (reviewed in reference 30
). Recently, we have shown that the overexpression of the transmembrane domains of the Sindbis virus E2 and E1 envelope glycoproteins induces apoptosis in AT3 cells (15
). In addition, coiled-coil proteins such as Beclin may play a role in linking membrane signal transduction events with the cytoskeleton. Thus, we speculate that Beclin, a protein which localizes to intracellular membranes, may be part of a complex with Bcl-2-like proteins that regulates signalling events initiated by Sindbis virus envelope glycoproteins in the endoplasmic reticulum.
Many similarities exist between the findings of the present study and our previous work examining the effects of Bcl-2 overexpression on the natural history of Sindbis virus encephalitis. As observed with Beclin in this study, the overexpression of Bcl-2 in virally infected neurons has previously been shown to reduce CNS apoptosis, reduce CNS Sindbis virus replication, and reduce Sindbis virus mortality (17
). However, in the present study, we also identify one significant biologic difference between Bcl-2 and Beclin; namely, Beclin can exert protective effects against infection by a neurovirulent strain of Sindbis virus that is known to overcome the protective effects of Bcl-2. Ubol et al. demonstrated that a histidine substitution for wild-type glutamine at position 55 of the Sindbis virus E2 envelope glycoprotein was sufficient to enable the virus to kill cells expressing Bcl-2 (41
). Similarly, we have previously found that Bcl-2 expressed in a strain of Sindbis containing a glutamine at position 55 (strain 633) protects against fatal encephalitis (17
), but in the present study, we found that Bcl-2 expressed in a strain of Sindbis containing a histidine at position 55 (strain TE12) confers no protection against fatal disease. Yet, when expressed in an E2-55 histidine-containing background strain of Sindbis virus, Beclin confers significant protection. Thus, the mutation in E2, which confers neurovirulence, can counter the protective effects of Bcl-2 but not of Beclin.
Several explanations have been proposed to explain the ability of the E2-55 histidine mutation to confer resistance to Bcl-2 protection against Sindbis virus-induced death. Ubol et al. suggested that the neurovirulent mutation in E2 might somehow alter either a direct interaction between E2 and Bcl-2 in the endoplasmic reticulum or a possible effect of Bcl-2 on E2 protein folding or posttranslational modifications (41
). More recently, Grandgirard et al. raised the possibility that the mutation in E2 facilitates the access of caspases to Bcl-2 and subsequent triggering of Bcl-2 cleavage (10
). If viral activation of caspase-induced cleavage of Bcl-2 does prove to be an important mechanism of neurovirulence of Sindbis virus strains containing E2-55 histidine, our data suggest that Beclin may serve as an important host defense factor against this strategy of neurovirulence. This hypothesis is based on the ability of Beclin, a Bcl-2-interacting protein, to protect against neuronal death induced by the TE12 strain of Sindbis virus.
Our findings suggest that the antiviral effects of Beclin observed in Sindbis virus-infected mouse brains may be exerted at a stage of viral replication after viral RNA synthesis. At 2 days after chimeric virus infection, Beclin overexpression was associated with a 50-fold reduction in viral titers but no reduction in the number of viral RNA-positive cells. This finding suggests a block or abnormality at the level of either translation of the Sindbis virus structural proteins, posttranslational modifications of viral glycoproteins, virus assembly, or budding. This observation contrasts with previous studies examining the effects of Bcl-2 on infection with a related alphavirus, Semliki Forest virus (34
). On the basis of in situ hybridization studies for viral RNA and immunostaining for viral proteins, Scallan et al. concluded that bcl-2
functions at an early stage of the virus life cycle, either entry, pretranscriptional events, or transcription, to inhibit Semliki Forest virus replication (34
). The discrepancy between the findings of Scallan et al. and those reported in the present study may reflect fundamental differences between the antiviral effects of Beclin and Bcl-2, differences between Sindbis virus and Semliki Forest virus, or differences between the cell types infected. Alternatively, it is possible that Bcl-2 and/or Beclin act at multiple overlapping stages of the alphavirus life cycle and that different experimental designs in the two studies uncovered effects on different phases of replication. Given the lack of antiviral activity of Beclin lacking the Bcl-2-binding domain, it seems likely that the mechanisms by which Beclin and Bcl-2 inhibit Sindbis virus replication in mouse brain are similar.
The results of the present study do not permit us to evaluate whether the beneficial effects of Beclin on Sindbis virus-induced mortality are a consequence of antiviral effects, antiapoptotic effects, or a combination of both. It is not known whether Beclin prevents neural cell death solely as a consequence of reducing Sindbis virus replication in neurons or whether Beclin exerts antiapoptotic effects independently of its effects on viral replication. Further studies examining the effects of Beclin on apoptotic death in response to nonviral stimuli will be necessary to determine whether Beclin, like Bcl-2, functions as a general apoptosis inhibitor. If so, structure-function analyses of Beclin may be helpful to map domains important for antiviral and antiapoptotic function and to identify whether these two properties are interrelated or independent.
Previously, based on our observations with Bcl-2 and Sindbis virus infection, we postulated that molecular links may exist between cellular regulation of viral replication and cellular regulation of apoptotic death in neurons (17
). Our findings that Beclin has antiviral activity and that Beclin interacts with the cell death regulator Bcl-2 further support this hypothesis. In dividing cells, inhibition of virus-induced cell death by virally encoded cell death inhibitors such as p35 (7
) and adenovirus E1B can increase viral replication (3
). However, in nondividing, terminally differentiated cells such as neurons, genetic pathways may exist which permit preservation of the life of the cell, without the adverse consequence of increased total viral burden for the organism. Genes such as bcl-2
that are normally expressed in mammalian neurons may be important components of such pathways.