All known positive-strand RNA viruses replicate their genomes in association with membranous structures that are formed after the synthesis of viral proteins in infected cells by remodeling membranes from existing intracellular organelles. Different viruses target different organelle membranes (e.g., endoplasmic reticulum [ER], Golgi, endosomes, and mitochondria) and generate different morphological structures on which the replication complexes assemble (37
Among the Picornavirus
family members, RNA replication complexes from poliovirus-infected HeLa cells have been the best studied. Heterogeneously sized, vesicle-like structures that cluster in the perinuclear space in infected cells were observed by electron microscopy and described more than 40 years ago. Viral RNA replication occurs on the cytosolic surfaces of the vesicles, which aggregate into clusters (7
). Little is known about how these structures are formed, although some recent observations have suggested that components of the cellular secretory and/or autophagy pathways are involved (24
The process of membrane traffic begins at the ER, where polio proteins appear to be synthesized. Rust et al. (36
) have visualized, by three-dimensional reconstruction of serial confocal microscope sections, poliovirus protein 2B sequences, and presumably other viral proteins, budding from multiple sites on the ER and showed extensive colocalization of the 2B sequences with COPII coatamer protein. Although it has not been determined which polio protein sequences are directly responsible for initiating the COPII-dependent budding process, it is generally believed that the P2 proteins 2C, 2BC, or precursors thereof, are required (2
). Normally, the COPII machinery establishes a membrane flow from the ER to the Golgi complex. However, poliovirus replication vesicles do not fuse with the Golgi complex; indeed, the Golgi complex appears to disassemble in poliovirus-infected cells, presumably because retrograde membrane traffic from the Golgi continues while anterograde traffic is rerouted to form the viral replication complexes.
A complicating observation is that polio RNA replication is inhibited by the fungal antimetabolite, brefeldin A (BFA) (18
), whereas neither COPII-dependent traffic nor autophagous vesicle formation is known to be sensitive to this drug. Instead, BFA is known to inhibit the activation and function of some members of the small GTPase family, Arf, by interacting with specific guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive GDP-bound form to its active, GTP-bound form (30
). When activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating domains competent for cargo transport (29
). Thus, the Arf GTPases play a central role in regulating membrane dynamics and protein transport. The recruitment of Arfs to their target membranes is mediated by a diverse group of GEFs, all of which share a Sec7 domain necessary for guanine nucleotide exchange (23
). All Arf GEFs are peripherally associated membrane proteins either tethered to membranes through a pleckstrin homology domain that interacts with specific phosphoinositides or through interactions with specific membrane-bound proteins that are not well characterized. The latter group includes the high-molecular-weight GEFs GBF1, BIG1, and BIG2, which are sensitive to BFA (14
). The binding of specific GEFs to various membrane sites confers specificity to the recruitment and activation of specific Arfs, serving to regulate different steps in the membrane trafficking pathway (4
The sensitivity of poliovirus RNA replication to BFA suggested that, in addition (or subsequent) to the COPII-dependent budding of vesicles from the ER, an Arf-dependent membrane trafficking step may be required for polio replication complex formation. For example, Gazina et al. (21
) reported that β-COP, a component of the coatamer COPI, whose recruitment to membranes is regulated by Arf1, localizes to the membranous replication complexes in cells infected by echovirus 11, a member of the Enterovirus
genus whose growth, like that of poliovirus, is sensitive to BFA. Arf1 was not present on vesicles associated with the replication complexes of picornaviruses such as encephalomyocarditis virus, whose growth is resistant to BFA.
Previously, we demonstrated that Arf1 proteins colocalize with the newly formed poliovirus replication complexes in infected cells and that some members of the Arf family are recruited to cellular membranes by poliovirus proteins synthesized in HeLa cell extracts that support the complete translation and/or replication of viral RNA in vitro (5
). These extracts have been used extensively to investigate many aspects of viral protein synthesis and processing, RNA replication, and assembly of infectious virions (3
), and we have shown previously that viral RNA synthesis, but not translation, is dependent upon the presence of intact membranes in the extracts (19
). Recruitment of Arf to the membranes was induced independently by two viral proteins from the P3, but not the P2, region: 3A and 3CD.
We show here that the two viral proteins, 3A and 3CD, independently recruit different Arf GEFs (GBF1 and BIG1/2, respectively) to membranes. These GEFs then are responsible for Arf activation and translocation to these membranes. The requirement for these specific BFA-inhibited GEFs explains the sensitivity of virus growth to BFA, which can be rescued by overexpression of GBF1.