Coxsackievirus Infection Induces Autophagy-Like Vesicles and Megaphagosomes in Pancreatic Acinar Cells In Vivo

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.Macroautophagy—henceforth referred to as autophagy—is an intracellular process that is important for cellular differentiation, homeostasis, and survival. Through autophagy, long-lived cytosolic proteins and organelles become encapsulated within double-membraned vesicles, called autophagosomes, which fuse with lysosomes to facilitate degradation of protein and cellular organelles and to promote nutrient recycling/regeneration. Autophagy plays a key role in the host immune response to infection by viruses, bacteria, fungi, and parasites (reviewed in references 10 and 62). Within virus-infected cells, whole virions and/or viral proteins and nucleic acids are captured inside autophagosomes and degraded (following lysosomal fusion) through the process of xenophagy. Moreover, autophagosome fusion with the endosomal/lysosomal pathway facilitates Toll-like receptor recognition of viral materials and delivers endogenous cytosolic viral proteins to the major histocompatibility complex (MHC) class II antigen presentation pathway, which in turn may help to trigger activation of innate immunity (and type I interferon production) and promote antigen presentation to virus-specific CD4⁺ T cells (reviewed in references 9, 41, 44, 47, 72, and 90). A recent study has shown that autophagy is also involved in the processing and presentation of MHC class I-restricted viral epitopes (13).Given the importance of autophagy in antiviral immunity, it is perhaps not surprising that viruses have evolved mechanisms to evade and/or subvert this pathway (reviewed in references 9, 11, 14, 35, 37, 60, 61, and 77). Several members of the herpesvirus family, most notably herpes simplex virus type 1, inhibit autophagy within an infected cell and encode proteins that block and/or target intracellular signaling pathways that regulate autophagy (reviewed in references 60 and 61). However, some viruses not only evade autophagy but also appear to take advantage of the process; several RNA viruses induce autophagy and exploit the pathway during their replication (1, 12, 15, 31, 40, 43, 76, 93, 96). Viruses belonging to the Picornaviridae family and the Nidovirales order replicate their genomes on double-membraned vesicles that resemble autophagosomes; these vesicles are notably smaller in size than cellular autophagosomes and are decorated with proteins derived from the autophagic pathway (19, 21, 31, 37, 67, 68, 71, 92). Viral proteins encoded by poliovirus and equine arterivirus can trigger the formation of these autophagy-like vesicles (79, 80), and the expression of a single poliovirus protein, 2BC, is sufficient to induce lipidation of the host autophagy protein light chain 3 (LC3), encoded by the Atg8 gene (87). Taken together, these studies suggest that some viruses subvert the autophagy pathway to generate double-membraned vesicles that provide a surface for RNA replication (8, 37, 88). In addition, these vesicles may permit newly formed virions to escape from infected cells via a nonlytic route (36, 85).Although studies have demonstrated that the autophagic pathway may play an important role in virus infection in vitro, either to promote or to restrict viral replication, we are just beginning to appreciate and understand the function and effects of autophagy for virus infections in vivo. Autophagy acts in an antiviral fashion to limit tobacco mosaic virus replication and programmed cell death in plants (46), to prevent a pathogenic infection with vesicular stomatitis virus in flies (73), and to protect against fatal encephalitis in Sindbis virus- or herpes simplex virus type 1-infected mice (45, 59, 63). Nonetheless, to date there is a dearth of in vivo studies; animal models of virus infection are needed in order to better define the antiviral role of autophagy in vivo (41, 62). In addition, studies that address the role of viral subversion of autophagy in vivo are warranted. Does this process occur within infected animals, and is it required for viral replication in particular cell types or for viral pathogenesis? Recent studies have shown that autophagy not only promotes the replication of hepatitis B virus and enterovirus 71 in vitro but also may be induced by infection in vivo, potentially to benefit the virus rather than the host (28, 78).Type B coxsackieviruses (CVBs) are members of the Picornaviridae family and Enterovirus genus and, as such, are closely related to polioviruses. CVBs are important human pathogens that often induce severe acute and chronic diseases and cause morbidity and mortality (69, 91). CVBs are the most common cause of infectious myocarditis (38, 82) and frequently trigger pancreatitis and aseptic meningitis (7, 16, 29, 51). Tissue culture studies (93) have shown that CVB type 3 (CVB3) promotes LC3 conversion and autophagosome accumulation in virus-infected cells in vitro and that modulation of the autophagic pathway (using chemicals or small interfering RNA-mediated knockdown) to enhance or dampen autophagy results in an increase and a decrease, respectively, in viral protein expression and/or viral titers; however, the reported changes in viral titers were modest (2- to 4-fold). In the present study, we examine whether CVB3 activates the autophagic pathway in vivo, specifically in pancreatic acinar cells, which are a natural primary target for this virus. Using a mouse model of CVB3 infection, which faithfully recapitulates most aspects of CVB disease in humans, we demonstrate that this virus triggers LC3 conversion and also modulates other components of the autophagy machinery. In addition, using a recombinant CVB3 (rCVB3) that expresses Discosoma sp. red fluorescent protein (DsRed-CVB3), we identify virus-infected cells in situ and show that CVB3 infection increases autophagosome abundance in vivo. Lysosomal-associated membrane protein 1 (LAMP-1) immunostaining confirmed that amphisomes are generated in virus-infected cells but that autophagic flux was not substantially enhanced as the infection progressed; rather, there appears to be a substantial blockade in fusion with lysosomes. Finally, transmission electron microscopy (TEM) ultrastructural analysis of the infected pancreas confirmed that double-membraned autophagy-like vesicles as well as very large autophagic compartments (for which we have coined the term “megaphagosomes”) were generated in acinar cells following virus infection. Overall, these data provide compelling evidence that CVB3 induces autophagy in vivo and suggest that this picornavirus may subvert this process in a mammalian host.