Role of Microtubules in Extracellular Release of Poliovirus

Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigenprocessing pathways have been reported, little is known about their relative contributions to global CD4؉ T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4 ؉ T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4؉ response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen. The activation of CD4ϩ T cells depends upon their recognition of peptides (epitopes) associated with major histo compatibility class (MHC) class II molecules. Conventionally, peptide generation involves the degradation of exogenous (extracellular) antigens in the endosomal network by multiple mechanisms, including unfolding, reduction, and proteolytic degradation. It is now clear that epitopes derived from endogenous antigens (synthesized by the cell) also can be presented on MHC class II molecules (12,22,24,38,49,50,60,66). Indeed, endogenous antigen expression appears to be an absolute requirement for the presentation of some epitopes. For example, the UV inactivation of A/PR8/34 influenza virus (PR8) or treatment of infected antigen-presenting cells (APCs) with protein synthesis inhibitors prevents the presentation of the NA79 epitope (12). Similar observations have been made for an epitope derived from influenza matrix protein 1 (24) and an epitope derived from the MHC class I H2-L d molecule (39).Numerous studies now have demonstrated that endogenous antigens can gain access to MHC class II loading compartments via a variety of intracellular pathways (4,38,49,57,66,67). Perhaps the most straightforward route is autophagy, in which cytosolic proteins are delivered to the lysosome via several different mechanisms. Chaperone-mediated autophagy results in the delivery of cytosolic proteins directly to the lysosome based upon the recognition of a KFERQ pentapeptide motif within the target protein, and it was shown to be responsible for the presentation of an epitope derived from glutamate decarboxylase (72). Although 30% of cytosolic proteins contain this motif (70)...

Section: Discussionmentioning

confidence: 99%

Functional Macroautophagy Induction by Influenza A Virus without a Contribution to Major Histocompatibility Complex Class II-Restricted Presentation

Comber

Robinson

Siciliano

et al. 2011

“…BTV association with vesicles in insect cells has not been previously reported. However, other nonenveloped viruses have been shown to be vesicle associated; Peruvian horse sickness virus, another member of the genus Orbivirus, associates with vesicles in mosquito-derived C6/36 cells (1), and for both the parvovirus minute virus of mice and the on May 10, 2018 by guest http://jvi.asm.org/ picornavirus poliovirus, egress has also been shown to be mediated by vesicles (24). These data suggest that while the N terminus of NS3 is not essential for virus growth in insect cells (as shown by growth of BTVM14), it is nonetheless involved in the trafficking of virus to exit the cell.…”

Section: Discussionmentioning

confidence: 99%

Interaction of Calpactin Light Chain (S100A10/p11) and a Viral NS Protein Is Essential for Intracellular Trafficking of Nonenveloped Bluetongue Virus

Celma

Roy

2011

Bluetongue virus (BTV), a member of the Reoviridae family, is an insect-borne animal pathogen. Virus release from infected cells is predominantly by cell lysis, but some BTV particles are also released from the plasma membrane. The nonstructural protein NS3 has been implicated in this process. Using alternate initiator methionine residues, NS3 is expressed as a full-length protein and as a truncated variant that lacks the initial 13 residues, which, by yeast-two hybrid analyses, have been shown to interact with a cellular trafficking protein S100A10/p11. To understand the physiological significance of this interaction in virusinfected cells, we have used reverse genetics to investigate the roles of NS3 and NS3A in virus replication and localization in both mammalian and insect vector-derived cells. A virus expressing NS3 but not NS3A was able to propagate in and release from mammalian cells efficiently. However, growth of a mutant virus expressing only NS3A was severely attenuated, although protein expression, replication, double-stranded RNA (dsRNA) synthesis, and particle assembly in the cytoplasm were observed. Two of three single-amino-acid substitutions in the N-terminal 13 residues of NS3 showed phenotypically similar effects. Pulldown assay and confocal microscopy demonstrated a lack of interaction between NS3 and S100A10/p11 in mutants with poor replication. The role of NS3/NS3A was also assessed in insect cells where virus grew, albeit with a reduced titer. Notably, however, while wild-type particles were found within cytoplasmic vesicles in insect cells, mutant viruses were scattered throughout the cytoplasm and not confined to vesicles. These results provide support for a role for the extreme amino terminus of NS3 in the late stages of virus growth in mammalian cells, plausibly in egress. However, both NS3 and NS3A were required for efficient BTV growth in insect cells.In recent years, accumulating evidence about the nonlytic release of nonenveloped viruses has contradicted the view that the release of these viruses is a passive process due to cell lysis. Studies on small nonenveloped viruses such as simian virus 40 (8) and poliovirus (25) have indicated that these viruses may exit from the infected cells nonlytically. Our lab has also previously provided strong evidence that bluetongue virus (BTV), a member of the Reoviridae family, exits the infected cells not only by cell lysis but also by newly synthesized particles budding from the plasma membrane early in infection (16). Thus, the egress of nonenveloped viruses appears to be a complex process and requires further investigations.BTV is vectored by insect Culicoides spp. to ruminants, causing disease in vertebrate hosts (sheep, cattle, and goats) that has considerable economic impact. Thus, BTV has the ability to replicate in both insect and mammalian hosts. In cell culture, mammalian cells infected with BTV exhibit strong cytopathic effects (CPE) after a few hours of infection. In contrast, no obvious CPE can be visualized in insect vector cell c...

“…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.…”

mentioning

confidence: 98%

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

Kemball¹,

Alirezaei²,

Flynn³

et al. 2010

145

182

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, longlived 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 ϩ 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 prote...