Potential subversion of autophagosomal pathway by picornaviruses

The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation.However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200- 400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.     

Potential subversion of autophagosomal pathway by picornaviruses

The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation. However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200-400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to reduce viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.     

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

Infection of mammalian cells with several positive-strand RNA viruses induces double-membraned vesicles whose cytosolic surfaces serve as platforms for viral RNA replication. Our recent publication (Jackson et al., PLoS Biology 3: 861-871, 2005) chronicled several similarities between poliovirus-induced membranes andautophagosomes, including induced co-localization of GFP-LC3 and LAMP1. Occasionally, the cytosolic lumen of these structures also contains viral particles; this likely results from wrapping of cytosol, which can contain high viral concentrations late in infection, by newly formed double membranes. Interestingly, RNAi treatment to reduce LC3 or Atg12p concentrations reduced yields of extracellular virus even more than intracellular virus. It is often assumed that exit of non-enveloped viruses such as poliovirus requires cell lysis. However, we hypothesize that autophagosome-like double-membranes, which can become single-membraned upon maturation, provide a long-sought mechanism for the observed non-lytic release of cytoplasmic viruses and possibly other cytoplasmic material resistant to the environment of maturing autophagosomes.     

Topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm

Infection of mammalian cells with several positive-strand RNA viruses induces double-membraned vesicles whose cytosolic surfaces serve as platforms for viral RNA replication. Our recent publication (Jackson et al. PLoS Biol 2005; 3:861-71) chronicled several similarities between poliovirus-induced membranes and autophagosomes, including induced co-localization of GFP-LC3 and LAMP1. Occasionally, the cytosolic lumen of these structures also contains viral particles; this likely results from wrapping of cytosol, which can contain high viral concentrations late in infection, by newly formed double membranes. Interestingly, RNAi treatment to reduce LC3 or Atg12p concentrations reduced yields of extracellular virus even more than intracellular virus. It is often assumed that exit of non-enveloped viruses such as poliovirus requires cell lysis. However, we hypothesize that autophagosome-like double-membranes, which can become single-membraned upon maturation, provide a long-sought mechanism for the observed non-lytic release of cytoplasmic viruses and possibly other cytoplasmic material resistant to the environment of maturing autophagosomes.     

Modification of cellular autophagy protein LC3 by poliovirus

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.     

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.     

Double-membraned Liposomes Sculpted by Poliovirus 3AB Protein

Infection with many positive-strand RNA viruses dramatically remodels cellular membranes, resulting in the accumulation of double-membraned vesicles that resemble cellular autophagosomes. In this study, a single protein encoded by poliovirus, 3AB, is shown to be sufficient to induce the formation of double-membraned liposomes via the invagination of single-membraned liposomes. Poliovirus 3AB is a 109-amino acid protein with a natively unstructured N-terminal domain. HeLa cells transduced with 3AB protein displayed intracellular membrane disruption; specifically, the formation of cytoplasmic invaginations. The ability of a single viral protein to produce structures of similar topology to cellular autophagosomes should facilitate the understanding of both cellular and viral mechanisms for membrane remodeling.     

Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena.

Poliovirus infection leads to an increase of phospholipid synthesis and the proliferation of new membranes, giving rise to a great number of cytoplasmic vesicles in the infected cells. Viral RNA replication is physically associated with these newly-synthesized membranes. Cerulenin, an inhibitor of lipid biosynthesis, effectively blocks the growth of poliovirus in HeLa cells. The presence of cerulenin after virus entry prevents the synthesis of poliovirus proteins. However, if this antibiotic is added at later stages of the virus replication cycle, it has no effect on viral translation itself, nor on the proteolytic processing and myristoylation of poliovirus proteins. The synthesis of viral, but not cellular RNA is selectively inhibited by cerulenin. Analysis of the viral RNA made in poliovirus-infected cells by specific minus-or plus-stranded RNA probes suggests a selective blockade by cerulenin of plus-strand RNA synthesis. Finally, the synthesis of phospholipids and the proliferation of membranes does not take place if cerulenin is added to the culture medium. These findings indicate that continuous phospholipid synthesis is required for efficient poliovirus genome replication and provide new insights towards the understanding of the molecular events that occur during poliovirus growth.     

RNase L Triggers Autophagy in Response to Viral Infections

Autophagy is a programmed homeostatic response to diverse types of cellular stress that disposes of long-lived proteins, organelles, and invading microbes within double-membraned structures called autophagosomes. The 2′,5′-oligoadenylate/RNase L system is a virus-activated host RNase pathway that disposes of or processes viral and cellular single-stranded RNAs. Here we report that activation of RNase L during viral infections induces autophagy. Accordingly, infections with encephalomyocarditis virus or vesicular stomatitis virus led to higher levels of autophagy in wild-type mouse embryonic fibroblasts (MEF) than in RNase L-null MEF. Similarly, direct activation of RNase L with a 2′,5′-oligoadenylate resulted in p62(SQSTM1) degradation, LC3BI/LC3BII conversion, and appearance of autophagosomes. To determine the effect of RNase L-mediated autophagy on viral replication, we compared viral yields in wild-type and RNase L-null MEF in the absence or presence of either chemical inhibitors of autophagy (bafilomycin A1 or 3-methyladenine) or small interfering RNA (siRNA) against ATG5 or beclin-1. At a low multiplicity of infection, induction of autophagy by RNase L during the initial cycle of virus growth contributed to the suppression of virus replication. However, in subsequent rounds of infection, autophagy promoted viral replication, reducing the antiviral effect of RNase L. Our results indicate a novel function of RNase L as an inducer of autophagy that affects viral yields.     

Hijacking components of the cellular secretory pathway for replication of poliovirus RNA

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.     

Role of Microtubules in Extracellular Release of Poliovirus

Cellular autophagy, a process that directs cytosolic contents to the endosomal and lysosomal pathways via the formation of double-membraned vesicles, is a crucial aspect of innate immunity to many intracellular pathogens. However, evidence is accumulating that certain RNA viruses, such as poliovirus, subvert this pathway to facilitate viral growth. The autophagosome-like membranes induced during infection with wild-type poliovirus were found to be, unlike cellular autophagosomes, relatively immobile. Their mobility increased upon nocodazole treatment, arguing that vesicular tethering is microtubule dependent. In cells infected with a mutant virus that is defective in its interaction with the host cytoskeleton and secretory pathway, vesicle movement increased, indicating reduced tethering. In all cases, the release of tethering correlated with increased amounts of extracellular virus, which is consistent with the hypothesis that small amounts of cytosol and virus entrapped by double-membraned structures could be released via fusion with the plasma membrane. We propose that this extracellular delivery of cytoplasmic contents be termed autophagosome-mediated exit without lysis (AWOL). This pathway could explain the observed exit, in the apparent absence of cellular lysis, of other cytoplasmic macromolecular complexes, including infectious agents and complexes of aggregated proteins.Infection with lytic viruses, by definition, leads to the destruction of host cells. In fact, lysis often has been assumed to be the only mechanism by which nonenveloped viruses can exit infected cells, because usually they do not have access to the lumen of the host secretory pathway. Nevertheless, nonenveloped viruses have been observed in the extracellular milieu in the apparent absence of cell lysis. For example, hepatitis A virus, a small nonenveloped RNA virus of the Picornaviridae, can establish persistent infections with no visible cytopathic effect, yet it has been observed to spread from cell to cell in tissue culture and in infected liver (21) (reviewed in reference 24). Poliovirus, also a picornavirus, can lyse cells in tissue culture and in infected animals. However, it also may be able to spread nonlytically: persistent infections of tissue culture cells have been reported to be associated with abundant poliovirus in the cell medium (41, 45, 46). The interpretation of all of these experiments is plagued by the possibility that the lysis of a very few cells could go undetected yet lead to the presence of substantial virus in the extracellular medium. Some experimental support for a nonlytic cellular exit route for poliovirus has come from studies of infected polarized monolayers of Caco2 cells. Even when there was no apparent breach in the monolayer, based upon the maintenance of electrical resistance, progeny virions were observed in the medium facing the apical, but not the basolateral, surface of the intact monolayer (65). Similar findings were reported for simian virus 40, a double-stranded DNA virus that assembles in the nuclei of infected cells (13). These observations suggest that virus can be released nonlytically from the apical cell surfaces.What kinds of processes in infected cells could give rise to nonlytic exit? Infection is known to lead to dramatic changes in the morphology and physiology of host cells visible by light and electron microscopy, classically termed the cytopathic effect. Within 3 to 4 h, cells infected with poliovirus accumulate large numbers of membranous vesicles (8). Although the topology and origin of these membranes have been debated, the original micrographs of Dales et al. (14) and ultrastructural analysis performed by high-pressure cryofixation and freeze substitution to preserve membrane morphology (22, 54, 58) have shown that poliovirus-induced vesicles predominately are double-membraned structures with cytoplasmic contents. Single-membraned vesicles could be induced in mammalian cells and in Saccharomyces cerevisiae by the expression of poliovirus proteins 2C and 2BC in isolation (3, 12, 63), whereas both viral proteins 2BC and 3A were required to induce the formation of double-membraned vesicles (58). By 5 or 6 h, several hours before the lysis of most cells, infected cells begin to lose attachment to the substratum, and the cellular microtubule and intermediate filament networks collapse (17, 39). The cytoskeletal alterations are dramatic (29); curiously, however, cytoskeletal integrity was found to have no effect on the intracellular yield of poliovirus (16).The double-membraned vesicles that accumulate during poliovirus infection resemble, in many respects, double-membraned cellular autophagosomes, which envelop cytosolic contents to target them for autophagic degradation (reviewed in references 31, 34, 40, and 44). Poliovirus-induced vesicles, like autophagosomes, contain lipidated cellular protein LC3 and the late endosomal protein LAMP-1 (27, 58, 61). The reduction of the amounts of autophagy proteins Atg12 and LC3 via RNA interference treatment led to a decrease in the yield of intracellular virus (27), supporting the idea that components of the autophagy pathway are subverted by poliovirus to benefit viral growth. Prompted by the finding that the reduction of autophagy proteins LC3 and Atg12 decreased the amount of extracellular virus even more than intracellular virus (27), we have suggested that the viruses within the cytoplasmic lumena of double-membraned vesicles are released into the extracellular milieu (27, 33, 62). Here, we provide evidence that the association of virus-induced vesicles with the microtubule network can modulate the extracellular release of poliovirus. These findings strengthen the argument that the autophagosomal constituents contribute to the nonlytic viral release and reveal a potential pathway for its control.     

Intracellular topology and epitope shielding of poliovirus 3A protein

The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.     

Autophagosome supports coxsackievirus B3 replication in host cells

Recent studies suggest a possible takeover of host antimicrobial autophagy machinery by positive-stranded RNA viruses to facilitate their own replication. In the present study, we investigated the role of autophagy in coxsackievirus replication. Coxsackievirus B3 (CVB3), a picornavirus associated with viral myocarditis, causes pronounced intracellular membrane reorganization after infection. We demonstrate that CVB3 infection induces an increased number of double-membrane vesicles, accompanied by an increase of the LC3-II/LC3-I ratio and an accumulation of punctate GFP-LC3-expressing cells, two hallmarks of cellular autophagosome formation. However, protein expression analysis of p62, a marker for autophagy-mediated protein degradation, showed no apparent changes after CVB3 infection. These results suggest that CVB3 infection triggers autophagosome formation without promoting protein degradation by the lysosome. We further examined the role of the autophagosome in CVB3 replication. We demonstrated that inhibition of autophagosome formation by 3-methyladenine or small interfering RNAs targeting the genes critical for autophagosome formation (ATG7, Beclin-1, and VPS34 genes) significantly reduced viral replication. Conversely, induction of autophagy by rapamycin or nutrient deprivation resulted in increased viral replication. Finally, we examined the role of autophagosome-lysosome fusion in viral replication. We showed that blockage of the fusion by gene silencing of the lysosomal protein LAMP2 significantly promoted viral replication. Taken together, our results suggest that the host's autophagy machinery is activated during CVB3 infection to enhance the efficiency of viral replication.     

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.     

Autophagy is involved in influenza A virus replication

Autophagy is involved in the replication of viruses, especially those that perform RNA assembly on the surface of cytoplasmic membrane in host cells. However, little is known about the regulatory role of autophagy in influenza A virus replication. Using fluorescence and electron microscopy, we observed that autophagosomes can be induced and identified upon influenza A virus infection. The virus increased the amount of the autophagosome marker protein microtubule-associated protein light chain 3-II (LC3-II) and enhanced autophagic flux. When autophagy was pharmacologically inhibited by either 3-methylademine or wortmannin, the titers of influenza A virus were remarkably decreased. Viral reduction via autophagy inhibition was further confirmed by RNA interference, through which two different proteins required for autophagy were depleted. Noticeably, the compounds utilized had no marked effect on virus entry or cell viability, either of which might limit viral replication. Furthermore, alteration of cellular autophagy via pharmacological reagents or RNA interference impaired viral protein accumulation. Taken together, these findings indicate that autophagy is actively involved in influenza A virus replication.     

Poliovirus proteins induce membrane association of GTPase ADP-ribosylation factor

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication.     

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.     

Cellular origin and ultrastructure of membranes induced during poliovirus infection.

Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.     

Adenovirus's last trick: you say lysis, we say autophagy

The last stage of the adenovirus replication cycle, lysis, is considered not very efficient and remains poorly understood. Pathogen infection induces autophagy in eukaryotic cells. In the case of viruses, autophagy is a double-edged sword that can either facilitate or impede replication. On one hand, autophagy reduces the replication capability of the herpesviruses. On the other hand, the RNA virus poliovirus uses autophagosomes to form replication complexes. Recently we characterized the autophagy induced by the oncolytic adenovirus Delta-24-RGD in brain tumor stem cells. Late in the adenoviral infectious cycle, we observed remarkable upregulation of the Atg12-Atg5 complex and prominent autophagy. In addition, adenovirus-induced autophagy results in disruption of the cytoplasmic structure and the continuity of the cellular membrane. We speculate that adenoviruses induce autophagy to facilitate the release of viral progeny at the end of the infectious cycle. The substitution of 'autophagy' for 'lysis' is not just semantic. Because autophagy is a genetically programmed process and not a passive phenomenon, it immediately suggests interactions between adenovirus proteins and autophagy regulators. Understanding the mechanism underlying adenovirus-mediated autophagy should propel the development of novel vectors with enhanced capability to release viral progeny and, as a result, morepotent oncolytic effect.     

Poliovirus metabolism and the cytoskeletal framework: detergent extraction and resinless section electron microscopy.

The association of poliovirus metabolism with the cytoskeleton was investigated. Infected cells were extracted by using the nonionic detergent Triton X-100 in the physiological cytoskeleton buffer. The skeletal framework obtained was examined by transmission electron microscopy of resinless sections. The fibers of the framework were grossly distorted in infected cells. No virions or procapsids were seen but many virus-specific spheroidal bodies were associated with the framework. They had a diameter of 40 to 70 nm, were characterized by a dense core and a translucent periphery, and occurred in strings, often near the remnants of flattened vesicles. These spheres may correspond to virus-synthesizing bodies. The metabolism of poliovirus RNA was shown to be associated with the skeletal framework by pulse-labeling cells with [3H]uridine and measuring the RNA retained on the framework. 20S double-stranded RNA, a form of poliovirus RNA found only in the replication complex, was attached to the skeleton throughout a 60-min pulse-label. 35S single-stranded viral RNA, a form found in virions, in polyribosomes, and in the replication complex, appeared first on the framework but after a few minutes was also found in the soluble cytoplasmic phase, encapsidated in virions. In contrast to viral RNA, viral proteins exhibited a varied association with the skeletal framework. Viral proteins were pulse-labeled with [35S]methionine and chased with unlabeled methionine. Although all of the virus-specific proteins were found, to some extent, in the skeletal fraction, the derivatives of P2 (P2-X and P2-5) and a derivative of P3 (P3-2) showed a preferential association with the skeletal framework. Virions and procapsids, on the other hand, were not associated with the cytoskeleton; both they and their component proteins (P1-VP0, P1-VP1, P1-VP2, and P1-VP3) were found dominantly in the soluble cytoplasmic phase. The pathway of poliovirus assembly can be inferred from the above data. It is different from that found previously for the enveloped vesicular stomatitis virus and may be representative of encapsidated cytoplasmic virus assembly.