Topology of Double-Membraned Vesicles and the Opportunity for Non-Lytic Release of Cytoplasm

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Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.All positive-strand RNA viruses of eukaryotes replicate their genomes on cytoplasmic membranes, and infections are often accompanied by dramatic reorganization of those membranes. We and others have argued that one function of membrane localization of viral RNA replication proteins is to create twodimensional surfaces to promote the oligomerization of viral proteins (6, 31). Yet the detailed cell biology of the targeted membranes and the mechanisms by which they are altered differ greatly from virus to virus (40,53).Poliovirus infection rearranges intracellular membranes, creating membranous vesicles, 200 to 400 nm in diameter, that accumulate in the cytoplasm (9, 42, 47). Immunoelectron microscopy revealed that the cytoplasmic surfaces of these membranous vesicles, not the interior lumen, are the sites of viral RNA replication (1, 7). The specific morphology of these membranes has proven somewhat controversial, because they are rich in lipids (17,33) and their apparent morphology is therefore sensitive to the fixation method used in electron microscopy (43, 46). Using high-pressure cryopreservation methods, we have consistently observed double-membraned vesicles (42, 47), as did Dales et al. using more conventional fixation techniques (9). Double-membraned vesicular structures have also been associated with viral RNA replication complexes in cells infected with several other positive-strand RNA viruses, including equine arterivirus (37), murine hepatitis virus (16), and sudden acute respiratory syndrome-associated virus (15). Therefore, understanding the mechanism(s) by which viral proteins induce the formation of double-membrane vesicles is of interest for understanding the formation of RNA replication complexes of several kinds of RNA viruses.Several of ...

Section: Discussionmentioning

confidence: 99%

Modification of Cellular Autophagy Protein LC3 by Poliovirus

Taylor

Kirkegaard

2007

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149

“…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: 88%

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

Kemball¹,

Alirezaei²,

Flynn³

et al. 2010

141

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...

“…The replication of (ϩ)RNA viruses takes place on the cytosolic surfaces of various cellular organelles, such as the endoplasmic reticulum (ER) and mitochondria, whereas some viruses actively induce the formation of novel cytoplasmic vesicular compartments (12,16,23,24,31,53,54). The emerging picture is that the mechanism of genome replication and the functions of viral and host factors might be analogous to some extent among various (ϩ)RNA viruses in spite of their diverse genome organization and gene expression strategies.…”

mentioning

confidence: 99%

A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes

Wang

Stork

Nagy

2009

127

131

Plus-stranded RNA viruses coopt host proteins to promote their robust replication in infected hosts. Tomato bushy stunt tombusvirus (TBSV) is a model virus that can replicate a small replicon RNA in Saccharomyces cerevisiae and in plants. The tombusvirus replicase complex contains heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, which is required for TBSV replication. To dissect the function of Hsp70 in TBSV replication, in this paper we use an Hsp70 mutant (ssa1 ssa2) yeast strain that supports a low level of TBSV replication. Using confocal laser microscopy and cellular fractionation experiments, we find that the localization of the viral replication proteins changes to the cytosol in the mutant cells from the peroxisomal membranes in wild-type cells. An in vitro membrane insertion assay shows that Hsp70 promotes the integration of the viral replication proteins into subcellular membranes. This step seems to be critical for the assembly of the viral replicase complex. Using a gene-silencing approach and quercetin as a chemical inhibitor to downregulate Hsp70 levels, we also confirm the significance of cytosolic Hsp70 in the replication of TBSV and other plant viruses in a plant host. Taken together, our results suggest that cytosolic Hsp70 plays multiple roles in TBSV replication, such as affecting the subcellular localization and membrane insertion of the viral replication proteins as well as the assembly of the viral replicase.Plus-stranded RNA [(ϩ)RNA] viruses replicate efficiently in host cells by assembling their own replicase complexes, consisting of virus-and host-encoded proteins, the viral RNA, and host membranes (1,2,10,33,34,36,41,54,56,58). The replication of (ϩ)RNA viruses takes place on the cytosolic surfaces of various cellular organelles, such as the endoplasmic reticulum (ER) and mitochondria, whereas some viruses actively induce the formation of novel cytoplasmic vesicular compartments (12,16,23,24,31,53,54). The emerging picture is that the mechanism of genome replication and the functions of viral and host factors might be analogous to some extent among various (ϩ)RNA viruses in spite of their diverse genome organization and gene expression strategies. Also, most of the previously identified host factors are conserved genes, suggesting that (ϩ)RNA viruses might selectively target conserved host functions as opposed to species-specific factors. This strategy can help viruses to have a broader host range and to expand infections to new host species. Host factors are also key determinants of virus pathology, host-virus interactions, and the evolution of the virus.The tombusvirus replicase is among the best characterized for (ϩ)RNA viruses due to the development of in vitro assays and Saccharomyces cerevisiae as a model host (33,35,44,46,48,55). Replication of tombusvirus RNA depends on the virusencoded RNA-dependent RNA polymerase (p92 pol RdRp) and the p33 replication cofactor, the key protein in the recruitment of the viral RNA into replication (50). The two viral proteins, in...