As discussed, autophagy promotes normal cellular functioning through constitutive turnover of potentially harmful cytosolic contents, and can be upregulated under stress conditions to promote survival. In addition, the routine turnover of cellular proteins by autophagy may prevent their accumulation and aggregation. As long-lived, non-regenerating cells, neurons rely extensively on autophagy to recycle organelles and proteins; without such basal autophagy, neurodegeneration can ensue (Boland and Nixon, 2006
). Similarly, like cytosolic contents, neurons may also rely extensively on autophagy to remove cytoplasmic viral proteins or particles. In contrast to most cell types in the organism where viral clearance occurs largely through cytotoxic T lymphocyte killing of virally infected cells (Wong and Pamer, 2003
), the destruction of virally infected neurons would be detrimental to the host, perhaps creating a unique reliance on non-cytolytic mechanisms of viral clearance. Along these lines, previous studies have demonstrated different non-cytolytic mechanisms for clearing Sindbis and other viruses from neurons, including antibody-mediated and cytokine-mediated restriction of viral gene expression (Levine et al., 1991
; Kimura and Griffin, 2000
; Binder and Griffin, 2001
). Recent data with Sindbis virus and HSV-1 infections in mice raise the possibility that autophagy may represent a newly described non-cytolytic mechanism for clearing viruses from neurons (, right).
Beyond its role in viral clearance, it is possible that autophagy may exert other protective functions during infection with neurotropic viruses (, right). For example, another mechanism contributing to the protective effects of autophagy against viral CNS disease may be its role in promoting the survival of infected neurons. As noted above, decreased apoptosis is observed in the brains of mice infected with a Sindbis virus construct that expresses Beclin 1 and decreased neuronal death is observed in the brains of mice infected with a mutant HSV-1 virus that cannot inhibit Beclin 1-mediated autophagy. Besides direct cytoprotective effects in virally infected neurons, it is also possible that autophagy indirectly reduces neuronal death in virally infected brains by decreasing the total CNS viral burden and number of infected cells. Further, the homeostatic function of autophagy may not only help keep neurons alive, but also minimize neuronal dysfunction, by protecting neurons against endoplasmic reticulum (ER) and oxidative stress that occurs during infection and/or by facilitating the removal of protein aggregates and damaged organelles.
Two lines of evidence support the hypothesis that the autophagic machinery exerts antiviral activity directly in virally infected neurons. First, Beclin 1 overexpressed in neurons from a double-subgenomic Sindbis virus promoter is sufficient to protect against fatal encephalitis (Liang et al., 1998
). Second, HSV-1 ICP34.5 exerts its autophagy inhibitory activity in HSV-1-infected neurons (Orvedahl et al., 2007
). However, these observations do not preclude non-cell autonomous effects of neuronal autophagy. In addition to cell autonomous roles in infected cells of the CNS, autophagy may function to protect against viral infection at a tissue and organismal level by activating innate and adaptive immunity.
As noted above, it has been demonstrated that autophagy may deliver both cytosolic and exogenous antigens to MHC class II molecules (Schmid et al., 2007
; A. Iwasaki et al
., unpubl. data), and it is known that the CNS cell types, microglia and astrocytes, express class II molecules (Collawn and Benveniste, 1999
). Therefore, it is possible that autophagy may contribute to class II presentation of viral antigens by microglia or astrocytes during CNS infection. Likewise, it is possible that the recently demonstrated role for autophagy in IFN production in peripheral dendritic cells (Lee et al., 2007
) may be conserved in neurons, a cell type that is known to produce high amounts of IFN in response to Sindbis virus and other neurotropic viral infections (Delhaye et al., 2006
) (, right). Thus, autophagy may play an integral role as both a primary barrier to productive viral infection in neurons and in activating immune responses in neurons and other cells in the CNS such as microglia and astrocytes.
While the precise mechanisms by which neuronal autophagy protects against CNS viral infection are unclear, it seems likely that autophagy functions to restrict viral replication. Ultrastructural analysis reveals the presence of Sindbis virions and ICP34.5 deletion mutant HSV-1 virions within autophagosomes (Seay, 2005
; Levine, 2006
; Talloczy et al., 2006
). Moreover, metabolic labelling of viral proteins demonstrates increased rates of degradation in the ICP34.5 deletion mutant virus-infected cells compared with wild-type HSV-1 virus-infected cells (Talloczy et al., 2006
). These results suggest that virions are targeted for xenophagic degradation in vitro
, and studies in mice provide correlative evidence that xenophagic degradation also restricts viral replication of neurotropic viruses in vivo
(Liang et al., 1998
; Orvedahl et al., 2007
Although Beclin 1 binding is important for promoting HSV-1 replication and neurovirulence in vivo
, studies in atg5−/−
MEFs suggest that autophagy may play a lesser role in restricting HSV-1 replication in vitro
(Alexander et al., 2007
). While PKR deletion or mutation of the eIF2α phosphorylation site is sufficient to restore wild-type levels of replication in MEFs infected with HSV-1 lacking the ICP34.5 gene (Talloczy et al., 2006
deletion does not significantly increase the replication of this mutant virus in MEFs. These findings suggest that, while ICP34.5 does inhibit autophagy in vitro
, the primary determinant of efficient replication of HSV-1 in vitro
is ICP34.5-mediated regulation of translational arrest rather than autophagy. Alexander and Leib (2008)
speculate that the differences observed between the apparent effects of autophagy in restricting HSV-1 replication in vivo
and in atg5−/−
MEFs may be due to cell type-specific factors or differences between cell culture and in vivo
environments. These differences highlight the potential unique importance of autophagy in restricting viral replication in neurons, which may explain the requirement for some neurovirulent viruses (e.g. HSV-1) to evade the autophagy pathway.