Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.     

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.     

Nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, RNA templates, and polymerase activity

Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ～50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only "zippered" together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.     

In vivo self-interaction of nodavirus RNA replicase protein a revealed by fluorescence resonance energy transfer

Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.     

The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.     

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.     

Template RNA Length Determines the Size of Replication Complex Spherules for Semliki Forest Virus

The replication complexes of positive-strand RNA viruses are always associated with cellular membranes. The morphology of the replication-associated membranes is altered in different ways in different viral systems, but many viruses induce small membrane invaginations known as spherules as their replication sites. We show here that for Semliki Forest virus (SFV), an alphavirus, the size of the spherules is tightly connected with the length of the replicating RNA template. Cells with different model templates, expressed in trans and copied by the viral replicase, were analyzed with correlative light and electron microscopy. It was demonstrated that the viral-genome-sized template of 11.5 kb induced spherules that were ∼58 nm in diameter, whereas a template of 6 kb yielded ∼39-nm spherules. Different sizes of viral templates were replicated efficiently in trans, as assessed by radioactive labeling and Northern blotting. The replication of two different templates, in cis and trans, yielded two size classes of spherules in the same cell. These results indicate that RNA plays a crucial determining role in spherule assembly for SFV, in direct contrast with results from other positive-strand RNA viruses, in which either the presence of viral RNA or the RNA size do not contribute to spherule formation.     

A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids

We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-susceptible state. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.     

Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

Background

Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.

Results

Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).

Conclusion

Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.     

Host acyl coenzyme A binding protein regulates replication complex assembly and activity of a positive-strand RNA virus

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ～50% smaller but ～4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.     

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.     

DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.     

Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells

For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.     

Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates

Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2a(pol) has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a(pol), and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2a(pol), 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2a(pol). Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.     

Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes

Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.All positive-strand RNA viruses replicate their genomes in association with cellular membranes. The formation and activity of the membrane-bound replication complexes (RCs) can result in extensive alteration of membrane structures (11, 40, 48). Different viruses use different cytoplasmic membrane compartments as platforms for replication. Currently, there is only a limited understanding of how the virus-encoded and cellular proteins coordinate the formation of the replication-induced membrane structures. We address the mechanisms of membrane-bound replication with alphaviruses, particularly Semliki Forest virus (SFV). The alphaviruses comprise several human and animal pathogens, including the encephalitogenic alphaviruses (e.g., Western, Eastern, and Venezuelan equine encephalitis viruses) as well as the recently reemerging chikungunya virus, which belongs to the SFV clade of alphaviruses. During the past 5 years, chikungunya virus has caused more than 2 million infections and 500 deaths, and a new strain has spread throughout the areas surrounding the Indian Ocean (50). The alphaviruses use mosquitoes as intermediate hosts and transmission vectors, and at present no vaccines or antivirals are available to control these infections.The cytoplasmic replication of alphaviruses depends on the four viral nonstructural (ns) proteins, nsP1 to nsP4, which are all essential and act as a membrane-bound replication complex. The nsPs are translated from the viral positive-sense RNA genome as one large polyprotein. Cleavages catalyzed by the nsP2 moiety result in the release of the individual proteins. A large fraction of the synthesized nsPs is involved in genome replication and associates with membranes, but a sizable fraction dissociates and is distributed in different cellular compartments: nsP1 binds to the inner surface of the plasma membrane (PM); nsP2 is translocated into the nucleus; nsP3 seems to form aggregates in the cytoplasm; and most of the extra nsP4, the core RNA polymerase, is degraded by the proteasome. While the major enzymatic functions of the individual nsPs have been elucidated (21), little is known of how they function together in the replication machinery.As in other positive-strand RNA viruses, the RCs of alphaviruses are associated with altered intracellular membranes, which were first described in the late 1960s and early 1970s (13, 14, 18). In these early studies, it was shown that virus replication induces bulb-shaped membrane invaginations with a diameter of ∼50 nm, which were called spherules. The spherules were found on the limiting membranes of large cytoplasmic vacuoles, which were named virus-induced cytopathic vacuoles of type I (CPV-I). On rare occasions, the spherules were seen also at the PM. By electron microscopic (EM) autoradiography, it was also shown that the spherules both at the CPV-I and at the PM could be sites of RNA synthesis (18). Subsequently, Froshauer et al. (15) showed that CPV-I are positive for endosomal and lysosomal markers. Moreover, using EM, they showed that the inside of the spherule is connected to the cytoplasm by a pore from which electron-dense material (which the authors suggest to be the newly synthesized RNA) seems to diffuse into the cytoplasm.During the past decade, our group has addressed the biogenesis of the CPV-I. We demonstrated that the formation of the spherules did not require structural proteins (44) and, more recently, that all four nsPs were associated with the spherules together with newly formed RNA (labeled by bromouridine), strongly suggesting that they were the actual units of RNA replication (RCs) (28). We also suggested as one possibility that the spherules could first arise at the PM; subsequent endocytosis of the spherules could account for the formation of the CPV-I (28, 44). Of the four nsPs, only nsP1 has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the PM (45). NsP1 is a monotopic membrane protein; its affinity for membranes is dictated by an amphipathic alpha helix, located in the central region of the protein (4, 31). NsP1 has a specific affinity for negatively charged phospholipids, which could potentially account for its prevalent localization to the PM, where such lipids are enriched. Later we showed that the membrane binding of nsP1 through the amphipathic helix is essential for alphavirus replication (56).Several groups of positive-sense RNA viruses make spherules, which appear very similar to those made by the alphaviruses. However, for these virus groups, the spherules arise in different locations. For the well-characterized brome mosaic virus (BMV), a plant virus very distantly related to the alphaviruses, the spherules are seen in the endoplasmic reticulum (ER) adjacent to the nucleus (51). For the unrelated nodaviruses, the spherules are localized on mitochondrial surfaces (25). Recent models of the RCs of flaviviruses suggest that their replication complexes also resemble spherules (62). For the Flaviviridae, the RCs are found on the membranes of the secretory pathway.The aim of this study was to clarify the role of different membranes in the formation and maturation of alphavirus RCs, and particularly to test our hypothesis that the RCs (spherules) are formed at the PM and are internalized thereafter. By using confocal microscopy, live-cell imaging, and novel electron microscopic techniques, we demonstrate that the RCs of SFV undergo an unprecedented, highly dynamic trafficking between different cellular compartments. They are first detected at the PM, which serves as the major platform for spherule formation. A specific endocytic event results in the transfer of spherules to the limiting membrane of small cytoplasmic vesicles. Using pharmacological inhibitors, we have been able to block the internalization process, and we found that the exit of spherules from the PM is dependent on the activity of phosphatidylinositol3- kinase (PI3K). Following the intracellular dynamics associated with spherules in live cells, we show the contribution of actin and microtubule-based transport, as well as that of fusion events with preexisting acidic organelles, providing the first complete model for the biogenesis of the large static CPV-I, where spherules are found at later stages of infection.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

Genome-Wide Analysis of Host Factors in Nodavirus RNA Replication

Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking.     

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.     

Complex dynamic development of poliovirus membranous replication complexes

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as "vesicles" are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses.