In this study, we investigated the interactions between FHV protein A and intracellular membranes, an initial and important step in the assembly of viral RNA replication complexes. Based on our results we conclude that protein A is a lipid-binding protein with particular affinity for specific anionic phospholipids. Furthermore, both biochemical and genetic studies indicated that protease-accessible membrane proteins, and in particular the TOM complex components Tom20, Tom70, and Tom40, were not required for protein A interactions with the mitochondrial membrane. However, we cannot exclude a role for protease-resistant and possibly membrane-embedded non-TOM complex mitochondrial outer membrane proteins in FHV protein A-membrane interactions. Indeed, although we saw almost complete association of in vitro-translated protein A with intact mitochondria, we obtained only partial association with lipid extracts or liposomes, with the exception of 100% CL liposomes. Furthermore, changes in membrane-embedded protein conformation may have accounted for the dramatic temperature dependence we observed in protein A mitochondrial binding. Nonetheless, the results with protein A in this study are consistent with those published for some endogenous mitochondrial outer membrane proteins that contain an amino-terminal signal-anchor transmembrane domain that resemble the targeting sequence of FHV protein A, suggesting a potentially similar pathway for membrane insertion (27
). The published study with Mcr1 used mitochondrial import assays, similar to the system described in this manuscript, including the use of protease digestion, TOM receptor component deletion strains, and Su9-DHFR competition experiments, but did not explore lipid binding (27
). Further experiments with both cellular mitochondrial proteins and FHV protein A will be required to fully define this pathway.
The observation that protein A has lipid-binding capabilities is consistent with the well-described association between positive-strand RNA virus replication complexes and intracellular membranes (32
). Host membranes likely play multiple roles in viral RNA replication, which may include (i) serving as a scaffold for assembly of a macromolecular structure such as an RNA replication complex, (ii) shielding viral RNA replication intermediates such as double-stranded RNA from cellular innate antiviral pathways, and (iii) providing cofactors for optimal enzymatic activity of viral replicase proteins. These functions could be mediated by either protein or lipid constituents within particular organelle membranes. Thus far the predominant emphasis in the field has been placed on identifying either membrane-resident proteins or cytosolic proteins that become membrane associated upon viral RNA replication complex assembly (13
). The results presented in this report indicate that lipids, and in particular anionic phospholipids, may also play important roles in viral RNA replication complex assembly.
The speculative role of lipids in replication complex assembly is consistent with the observation that the replicase protein nsP1 from Semliki Forest virus, a positive-strand RNA virus that assembles its replication complexes on membranes derived from endosomes and lysosomes (52
), also binds anionic phospholipids (1
). FHV protein A binding to predominantly anionic phospholipids suggests that ionic forces may mediate in part the interactions with phospholipids. Indeed, for nsP1 the anionic phospholipid interaction domain was mapped to an amphipathic α-helix with a cluster of positively charged residues (1
). Sequence and predicted secondary structural analyses have revealed the presence of several similar amphipathic α-helices within the FHV protein A-coding region that may be involved in anionic phospholipid interactions (K. Stapleford and D. Miller, unpublished data). However, the selectivity of protein A for certain anionic phospholipids suggests that nonionic forces also contribute to protein A-lipid interactions.
The ability of protein A to interact with several different anionic phospholipids suggests a level of “promiscuity” that may help explain the robustness of FHV RNA replication in cells derived from multiple organisms from several kingdoms (24
) and the relative ease with which FHV RNA replication complexes can be retargeted to alternative intracellular membranes such as the ER (31
). However, the ubiquitous nature of anionic phospholipids and the substantial amounts of PI and PS present in many cellular membranes, including the ER (62
), indicate that net membrane charge cannot be the sole determinant of replication complex targeting. Nonetheless, protein A did not bind all anionic phospholipids, but rather showed preferential interactions with CL, PA, and PG, which are enriched in mitochondrial membranes (55
), suggesting that specific anionic phospholipids can influence FHV replication complex targeting. Furthermore, we cannot exclude a role for local charge clusters within membrane microdomains or the impact of protease-resistant endogenous membrane protein interactions with anionic phospholipids in the membrane-specific targeting of FHV RNA replication complexes.
The hypothesis that anionic phospholipids play targeting or structural roles in FHV RNA replication complex assembly is particularly interesting given the results with CL, which we identified as a significant interaction partner of protein A. CL is a cellular phospholipid whose distribution is almost entirely limited to the mitochondria (17
). Although the majority of CL is found in the inner mitochondrial membrane, it is also present in high localized concentrations in the outer membrane at contacts sites between inner and outer membranes (3
). One might speculate that FHV RNA replication complexes may initially assemble at or near these contact sites via interactions between protein A and CL. This proposed mechanism resembles the targeting of the proapoptotic promoter tBID to CL that can initiate cytochrome c
-mediated apoptotic cell death (10
). The potential similarity between protein A-CL and tBID-CL interactions is intriguing given the recent demonstration that FHV infection induces apoptosis in cultured Drosophila
), although the delayed onset of FHV-induced apoptosis until approximately 12 h postinfection suggests that a threshold of protein A-CL interactions may be required. In addition, it is possible that protein A-mediated disruption of the inner-outer membrane contact sites, an essential substructure needed to maintain mitochondrial shape (44
), may lead to some of the morphological alterations that are seen in mitochondria from yeast or Drosophila
cells that contain active FHV RNA replication complexes (30
). This is consistent with the observation that components of the endogenous mitochondrial fission and fusion machinery can localize to these contact sites (44
) and that disruption of the normal fission-fusion processes can lead to abnormal mitochondrial morphology (36
) that also resemble mitochondria in cells with active FHV RNA replication (29
). Furthermore, the dimeric nature of CL, which consists of four acyl chains attached to diphosphatidylglycerol, imparts a unique conical structure that favors a hexagonal HII
phase that may play a role in the membrane curvatures necessary to produce the spherules associated with FHV RNA replication complexes (22
Although the focus of this study was to identify host components involved in protein A-membrane interactions, an initial step in FHV replication complex assembly, we found that protein A translated in RRLs had RNA polymerase activity when provided with an excess of exogenous virion template RNA template (data not shown). However, we did not observe the FHV RNA replication complex activity that has been described with membrane preparations from FHV-infected Drosophila
) and replicon-expressing yeast (31
), which includes the production of single-stranded products, with in vitro-translated protein A in the presence of whole mitochondria or liposomes. The particularly difficult feat of de novo assembly of fully functional viral RNA replication complexes using a cell-free in vitro translation system has only been accomplished for a select few positive-strand RNA viruses, including poliovirus, which requires the use of uninfected mammalian cell extracts (33
), the plant pathogens tomato mosaic virus, brome mosaic virus, and turnip crinkle virus, which require the use of evacuolated plant cell extracts (21
), and tomato bushy stunt virus, which uses a related system that employs a yeast cell extract and purified recombinant viral proteins (39
). Further studies with FHV will be required to identify the optimal conditions under which fully functional viral RNA replication complexes can be formed in vitro, and both the results presented in this report and others (59
) indicate that the inclusion of specific phospholipids may be a particularly important aspect of these studies.
In summary, the studies presented in this report demonstrate that FHV protein A mitochondrial association and membrane insertion is mediated by a TOM complex-independent mechanism, similar to what has been seen previously for mitochondrial outer membrane signal-anchored proteins, and provide evidence for the importance of host membrane-specific phospholipids in positive-strand RNA virus replication complex assembly. Future in vitro and in vivo studies using this established host-pathogen system will give further insight into the role of phospholipids in membrane-specific targeting as well as the biochemical mechanisms involved in positive-strand RNA virus replication complex assembly and function.