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 two-dimensional surfaces to promote the oligomerization of viral proteins (6
). Yet the detailed cell biology of the targeted membranes and the mechanisms by which they are altered differ greatly from virus to virus (40
Poliovirus infection rearranges intracellular membranes, creating membranous vesicles, 200 to 400 nm in diameter, that accumulate in the cytoplasm (9
). Immunoelectron microscopy revealed that the cytoplasmic surfaces of these membranous vesicles, not the interior lumen, are the sites of viral RNA replication (1
). The specific morphology of these membranes has proven somewhat controversial, because they are rich in lipids (17
) and their apparent morphology is therefore sensitive to the fixation method used in electron microscopy (43
). Using high-pressure cryopreservation methods, we have consistently observed double-membraned vesicles (42
), 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 the features displayed by the vesicles induced during poliovirus infection are shared with cellular autophagosomes, double-membraned organelles that become degradative upon maturation, breaking down cytoplasmic proteins and organelles entrapped within their lumen. Originally identified as a process induced by cellular starvation, autophagy is now appreciated as a cellular response to a variety of stimuli. For example, the estrogen receptor agonist tamoxifen is a potent inducer of autophagy, indicating a role for the hormonal induction of autophagy (2
). In Saccharomyces cerevisiae
, formation and maturation of autophagosomes requires the function of many genes (reviewed in reference 52
). Human homologs of several yeast autophagy genes have recently been identified, including LC3, the human homolog of ATG8, and it is likely that much of the autophagic pathway has been evolutionarily conserved (22
). Many recent experiments have identified the autophagy pathway as a critical component of the innate immune response of plants and mammals (reviewed in references 10
). Therefore, we sought to discover the similarities and differences between the poliovirus-induced membranes and cellular autophagosomes and to investigate the mechanism of formation of the double-membraned vesicles induced by poliovirus and the role of the autophagy pathway during viral infection. Previous studies show that reduced function of the autophagy pathway resulted in reduced amounts of extracellular and intracellular poliovirus (20
), arguing that the autophagic pathway plays a positive role in the growth of both these positive-strand RNA viruses.
The formation and maturation of autophagosomes involve the stepwise acquisition of proteins from disparate cellular compartments (reviewed in references 28
). Nascent autophagosomes form either de novo or from the endoplasmic reticulum and comprise cellular cytoplasm surrounded by two lipid bilayers that fuse from a C-shaped intermediate (14
). Although relatively protein poor, they have been shown to contain a modified form of the autophagy protein LC3. Originally characterized as microtubule-associated protein light chain 3, or MAP-LC-3, LC3 protein is cleaved after synthesis by cellular protease Atg4, generating an 18-kDa species termed LC3-I (see Fig. ). When autophagy is activated, a series of covalent transfers links LC3 to Atg7, then to Atg3, and finally to phosphatidylethanolamine, generating a lipidated species termed LC3-II (Fig. ) (19
). This modification allows LC3 to become membrane associated, preferentially associating with the developing and newly formed autophagosomes (23
FIG. 1. Modification of LC3 protein during poliovirus infection. (A) Schematic representation of LC3 processing as it occurs during autophagy activation. LC3-I is the unmodified, cleaved form of LC3, while LC3-II is the lipid-conjugated species. (B and C) Human (more ...)
The genomes of poliovirus and other picornaviruses consist of a single, positive-stranded RNA. A single encoded polyprotein is processed by viral proteases to produce individual capsid proteins, the viral RNA-dependent RNA polymerase 3D, and several other multifunctional nonstructural proteins. Of the nonstructural proteins, three (2B, 2C, and 3A) associate with membranes when expressed in isolation; mutations in any of these three proteins lead to defects in RNA replication. The expression of stable protein precursor 2BC and protein 3A, in combination but not separately, was found to induce the formation of double-membraned vesicles that display biochemical markers and fractionate similarly to the vesicles induced during poliovirus infection (20
). Thus, should these membranes prove to derive directly from the autophagy pathway, these viral proteins will be among the few molecularly characterized inducers of this process.
We showed previously that, during poliovirus infection, a green fluorescent protein (GFP)-fused version of one of the three cellular LC3 proteins, LC3b, becomes associated with cellular membranes that also contain viral RNA replication proteins (20
). Here, we investigated the possibility that the targeting of LC3 to membranes results from its covalent modification, as occurs during autophagy. Further, we tested the possibility that expression of an individual viral protein is sufficient to accomplish this modification.