RNA replication associated with altered cellular membranes has been described for all positive-strand RNA viruses. Although extensively studied, the function of this interplay is still not completely understood. It is suggested that membranes would provide a structural framework for the RC, anchoring the process to a confined place as well as concentrating the essential components needed for replication. In addition, lipids seem to offer a functional platform for the replicase proteins as their enzymatic activities depend on membrane binding (as described above for nsP1) −reviewed in (Salonen et al., 2005; Miller and Krijnse-Locker, 2008)−. As also mentioned, RNA
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replication produces dsRNA intermediates that are strong activators of the innate immune responses against viral infections. Therefore, the altered membranes may protect the replication intermediates from host defence mechanisms. It has been shown that interference with the correct assembly of membrane-bound complex/induction of membrane modifications makes the viral RNA more sensitive to ribonucleases (Barajas et al., 2009).
Membrane binding and targeting of viral RCs seems to be mediated via different mechanisms. Many viruses use amphipathic helices to anchor their RCs to the membranes similarly to alphaviruses. One well studied example is BMV, a plant virus belonging to the alphavirus-superfamily. In a recent study, it was shown that BMV anchors its RC to the membranes of ER via an amphipathic helix in the middle of the 1a protein (methyltransferase and helicase). Mutations in the helix abolished the membrane binding of the RC and were lethal to virus replication in plant host (Liu et al., 2009). Amphipathic helix fused with GFP was able to mediate the membrane association of the fusion protein (Liu et al., 2009). However, a longer region, termed helix E, was necessary to target the GFP to ER membranes (den Boon et al., 2001).
HCV possesses several amphipathic helices in different replicase proteins. NS4B (a palmitoylated protein with functions in modulating host cell environment) has been shown to contain two amphipathic helices in the N-terminus and at least one membrane-associated helix in the C-terminus. In addition, NS4B has four transmembrane domains in the central part of the protein and two palmitoylation sites in the C-terminus. Therefore, NS4B represents a true integral membrane protein.
Mutations in the amphipathic helices in the N- or C-terminus have been shown to affect the membrane binding and functionality of the RCs (Gouttenoire et al., 2010).
Replicase component NS5A has an N-terminal amphipathic helix that is conserved throughout the HCV-related viruses and mediates the membrane binding of the protein (Brass et al., 2007). Fusion of the helix with GFP targets the protein to the ER indicating that the amphipathic helix contains the targeting signal to the ER (Brass et al., 2002). Exceptionally, HCV polymerase domain NS5B is also membrane-associated having a C-terminal transmembrane domain (Moradpour et al., 2004).
Additionally, other replicase proteins are membrane-associated as well, leading to the situation where almost all the domains of the RC have individual membrane binding motifs. Therefore the membrane association of the HCV RC seems far more complicated than in some other cases. Poliovirus RCs are attached to the cellular membranes via several replicase proteins similar to HCV. 2CATPase protein possesses a predicted amphipathic α-helix in the N-terminus that mediates the membrane binding of the 2BC polyprotein (Echeverri and Dasgupta, 1995). Fusion of the first 72 aa form 2CATPase with chloramphenicol acetyltransferase (CAT) was able to target the recombinant protein to the membranes of ER (Echeverri et al., 1998). 3A mediates the membrane association of 3AB polyprotein through a hydrophobic sequence in the C-terminus of the 3A (Towner et al., 1996). 3AB is shown to anchor the RC to the membranes as it recruits the polymerase 3DPol through the interactions with 3B (Fujita et al., 2007). From these examples it is apparent that membrane binding mechanism of the RCs might be rather complex and differ between different virus groups.
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The formation of membrane-bound RC including the mechanisms as well as the components involved in inducing membrane alterations is the other large research topic concerning membrane-associated replication. Today, many aspects of the assembly of the membranous RCs are still not completely understood and seem to vary between different viruses. For several virus groups it has been shown that protein components can induce membrane alterations similar to rearrangements seen during virus infection. Expression of BMV 1a protein induces the formation of spherule-like structures on ER membranes. Mutations in the amphipathic α-helix are able to alter these structures, changing their size or appearance (Liu et al., 2009). Interestingly, co-expression of high levels of polymerase domain 2a changes the morphology of membrane alterations causing the induction of karmellae-like stack (double-membrane ER layers) around the nucleus (Schwartz et al., 2004). These results highlight a very delicate and important interplay between 1a and polymerase in order to form functional RCs. Similar observations have been made with Poliovirus replicase proteins. Expression of 2BC or 2CATPase results in the proliferation of ER membranes but co-expression of 3A is required in order to induce double-membrane vesicles (DMVs) that are associated with autophagy protein LC3 (Taylor and Kirkegaard, 2008). Only one HCV replicase protein NS4B is needed to induce the membranous web (Egger et al., 2002). C-terminal domain has been implicated to be involved in the process, although it is not sufficient to form the web (Aligo et al., 2009). Coronaviruses induce double-membrane vesicles (DMVs) that are thought to be the early RCs and later larger assemblies termed convoluted membranes (CMs) and vesicle packets (VPs) are associated with DMVs in the perinuclear area (Knoops et al., 2008; Hagemeijer et al., 2009). At least three nsPs (nsP3, nsP4 and nsP6) have been shown to have transmembrane domains. Expression of membrane-bound nsPs seems to induce the formation of DMVs. Additionally nsP3 and nsP6 contain hydrophobic domains that are shown to mediate membrane binding but their exact role is not known. It is suggested that they might resemble amphipathic helices that are shown to be involved in membrane remodelling (Oostra et al., 2008).
In addition to viral proteins, host components have been shown to be involved in the formation of viral RCs and subsequent membrane alterations. Tombusviruses (e.g.
TBSV) induce spherule-like structures on the membranes of chloroplasts and peroxisomes. A recent study indicates that cellular endosomal sorting complex required for transport (ESCRT) proteins are necessary for spherule formation (Barajas et al., 2009). TMV replicates on the membranes of ER and host TOM1 and TOM2A are needed for membrane association and replication (Yamanaka et al., 2000;
Tsujimoto et al., 2003). Poliovirus replication is dependent on coat protein (COP) I functions and RCs associate with COPI-coated membranes (Gazina et al., 2002).
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