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Bromovirus-induced remodeling of host membranes during viral RNA replication
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ScienceDirect Bromovirus-induced remodeling of host membranes during viral RNA replication Arturo Diaz1 and Xiaofeng Wang2 With its high yield, small genome, and ability to replicate in the yeast Saccharomyces cerevisiae, Brome mosaic virus (BMV) has served as a productive model to study the general features of positive-strand RNA virus infection. BMV RNA is replicated in spherules, vesicle-like invaginations of the outer perinuclear endoplasmic reticulum membrane that remain connected to the cytoplasm via a neck-like opening. Each spherule contains the viral replicase proteins as well as genomic RNAs. Recent advances indicate that multiple interactions between the viral proteins with themselves, cellular membranes, and host factors play crucial roles in BMV-mediated spherule formation. These findings are probably applicable to other positive-strand RNA viruses and might potentially provide new targets for antiviral treatments. Addresses 1 Department of Biology, La Sierra University, Riverside, CA 92505, United States 2 Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech University, Blacksburg, VA 24061, United States Corresponding authors: Diaz, Arturo ([email protected]) and Wang, Xiaofeng ([email protected])
Current Opinion in Virology 2014, 9:104–110 This review comes from a themed issue on Special Section: Roseoloviruses Edited by Laurie Krug
http://dx.doi.org/10.1016/j.coviro.2014.09.018 1879-6257/Published by Elsevier B.V.
life cycle. Because of the limited coding capacity of positive-strand RNA viruses, the formation and function of the viral replication compartments require complex interactions between the viral proteins, viral genome, and co-opted host factors. Recent work has shown that cellular host proteins that either structurally induce and/or support membrane curvature are required to form the replication compartments [2,3,4]. Moreover, cellular lipid synthesis and appropriate lipid composition are essential for viral replication [5], implying that the membrane is an essential, functional component of the RNA replication compartments. Host membrane remodeling and the role of host genes in viral replication of other positive-strand RNA viruses have been summarized in the following reviews [1,3,6,7]. This review will focus on recent advances on the formation of the BMV-induced RNA replication complexes. Brome mosaic virus (BMV) is the type member of the Bromoviridae family and a representative member of the alphavirus-like superfamily of human, animal, and plant positive-strand RNA viruses. With its high yield, tripartite genome and ability to replicate in the yeast Saccharomyces cerevisiae, BMV has served as a productive model to study some of the general features of positive-strand RNA virus infection, including viral genomic RNA replication, gene expression, recombination, and virus–host interactions [8]. In the following sections we will summarize recent work that has enhanced our understanding of BMV replication complex assembly and structure, both of which are probably applicable to other positive-strand RNA viruses as seemingly diverse membrane rearrangements may represent topologically and functionally related structures.
General features of spherule formation Introduction The RNA replication compartments of positive-strand RNA viruses are mini-organelles that feature the close association of both viral proteins and host cellular components [1]. Viral RNA replication takes place in close association with cellular membranes and although the architecture of these compartments differs among viral families, they usually involve membrane vesicle formation or other membrane rearrangements. Cellular membranes provide a scaffold for the enrichment of the viral replication factors as well as exploited cellular proteins, provide an environment to protect the viral RNA from cellular defense mechanisms and help to separate and coordinate the various stages of the viral Current Opinion in Virology 2014, 9:104–110
BMV has three genomic RNAs and one subgenomic mRNA. RNA1 encodes the multifunctional replication factor 1a, which contains an N-proximal RNA capping domain that is separated from a C-terminal NTPase/RNA helicase-like domain by a short proline-rich sequence that may be a flexible spacer [9–12]. 2apol is encoded from RNA2 and contains a central polymerase domain and an N-terminal domain that interacts with the helicase-like domain of 1a [13,14]. Genomic RNA3 encodes the 3a movement protein, which is required for cell-to-cell spread in plants, while subgenomic RNA4 encodes for the coat protein [15]. In both yeast and plant cells, BMV RNA replication depends on the viral 1a and 2apol proteins and specific www.sciencedirect.com
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cis-acting RNA signals [16], generates a considerable excess of positive-strand to negative-strand RNA [17], and efficiently directs subgenomic RNA4 synthesis [17]. In yeast, in the absence of other viral factors, 1a induces 60–80 nm vesicular invaginations in the outer membrane of the perinuclear endoplasmic reticulum (ER), which are referred to as spherules [18] (Figure 1). Spherules are bounded by a single lipid bilayer and the interior of these vesicles is connected to the cytoplasm through a neck-like opening, probably providing a channel for ribonucleotide import and product RNA export [18]. Confocal microscopy shows that 1a accumulates in discrete ER patches that expand during the course of infection [19] while biochemical and immunogold labeling analyses suggest that there are hundreds of 1a’s per spherule and only a few copies of 2apol [18]. Consistent with this, 1a interacts with and recruits 2apol to the perinuclear ER [18,19,20]. The 1a-mediated recruitment and stabilization of genomic RNA appear to include two steps, a 1a-induced recruitment of RNA to the ER membrane followed by the translocation of the RNA into preformed spherules [12]. Moreover, incorporation of BrUTP shows nascent RNA synthesis occurs within the spherules [18] (Figure 1). However, modulating the relative levels and interactions between replication factors 1a and 2apol shifts the membrane rearrangements from spherular compartments to large and multilayer stacks of appressed double-membrane
layers that support RNA replication as efficiently as spherules [21]. The following two sections will summarize recent work that sheds light into the manner by which 1a interacts with itself, lipid membranes, and other viral components and the details by which it invaginates the ER membranes to induce spherule formation. Roles of Helix A
1a has no trans-membrane domain and fully resides on the cytoplasmic side of the ER membrane [22]. A small amphipathic a-helix, termed Helix A, is crucial for both 1a’s membrane association and 1a-induced membrane rearrangement as well as 1a-mediated recruitment of viral RNA templates and 2apol [22,23] (Figure 1). Genetic, biochemical, and NMR analyses showed that mutations within Helix A give rise to two classes of mutants with distinct properties. Class I mutants, which disrupt helix formation, significantly inhibit 1a membrane affinity and fail to induce spherule formation but are over twice as effective as wild-type (wt) 1a at stabilizing and recruiting 2apol to nonperinuclear ER membranes [23]. By contrast, Class II mutants interact with 2apol inefficiently but increase the frequency of 1a-induced spherules by 5-fold, although these compartments are 30% smaller in diameter than those induced by wt 1a [23] (Figure 2b). Correspondingly, Class I mutants fail to recruit the viral
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Interactions and host factors required for the formation of functional BMV RNA replication complexes. 1a and 2apol interact in the cytoplasm before membrane association. 1a associates with the outer ER membrane, where 1a–1a and 1a–membrane interactions, in conjunction with host factors, lead to the invagination of the ER membrane to form spherules. 1a-mediated recruitment of viral RNA templates occurs after the formation of spherules, followed by the synthesis and retention of negative-strand RNA (dashed black lines), and asymmetric synthesis and export of positive-strand progeny RNA (red lines). Host factors involved in maintaining proper membrane lipid composition around the spherule membranes and in generating and/or maintaining the virus-induced membrane rearrangements are shown. www.sciencedirect.com
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BMV-induced membrane rearrangements formed under various conditions. Representative electron micrographs of spherules made by (a) wt 1a or (b) 1a with a Class II mutation. (c) Hexagonal arrays induced by the N-terminal capping domain of 1a. Spherules made in (d) ole1w, (e) acb1D, or (f) rtn1D mutant yeast strains. Arrows point out individual spherules. Nuc: nucleus; Cyto: cytoplasm. Scale bars, 200 nm. Electron micrographs are reproduced with permission from [4,23,24,30,35] and [4], respectively.
RNA while Class II mutants enhance RNA recruitment to even higher levels than wt 1a, further showing that RNA recruitment and protection by 1a strongly correlate with 1ainduced spherule formation [23]. Current Opinion in Virology 2014, 9:104–110
These results imply that the 1a–2apol interactions occur before spherule formation and that 1a–2apol interaction and 1a-induced spherule formation are sequential and perhaps antagonistic [23] (Figure 1). Moreover, the fact www.sciencedirect.com
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that Class II mutants alter spherule sizes suggests that 1a– membrane and/or 1a–1a interactions influence the diameter of these structures. 1a–1a interactions and multimerization
To further identify the determinants of spherule formation, the N-terminal capping and C-terminal helicaselike domains of BMV 1a were separately examined. Although both the capping and helicase domains individually multimerize, neither domain on its own induces spherule formation [24]. In vivo, the 1a capping domain forms membrane-linked hexagonal lattices of tubules or stacked rings and induces layering of ER double membranes while the helicase domain fails to associate with membranes [24] (Figure 2c). When expressed in trans, the capping domain fails to recruit the helicase domain to ER membranes, consistent with prior yeast two-hybrid results implying that the capping and helicase domains interact intramolecularly within full-length 1a but do not significantly interact intermolecularly between two 1a’s [25]. Moreover, when co-expressed, the capping domain recruits full-length 1a into hexagonal tubule lattices and inhibits 1a’s ability to form spherules and support RNA replication [24]. The results suggest at least three different interactions between 1a proteins: intramolecular interactions between the capping and helicase-like domains, intermolecular interactions between capping domains, and independent intermolecular interactions between helicase-like domains (Figure 1). It is possible that all of these contacts form simultaneously such that 1a molecules contact one another at multiple interfaces or that some interactions or multimeric states may be specific to certain 1a functions. The sum of the intramolecular and inter-molecular interactions appears to lead not only to the emergence of an extensive multimeric 1a lattice but also to the invagination of the perinuclear ER membrane that results in the generation of spherules. Disrupting any of those interactions inhibits spherule formation, for example, 1a-induced membrane rearrangements shift from spherules to appressed double-membrane layers when the helicase interacting 2apol is overexpressed [21], probably by interfering with helicase–helicase interactions between adjacent 1a proteins. 1a’s strong membrane association and asymmetric insertion of Helix A into the lipid bilayer to generate local membrane curvature [22,23], its ability to self-interact [24,25], and its high multiplicity within spherules [18] suggest that 1a forms a shell lining to induce and maintain the vesicular replication compartments (Figure 1). In order to form such a membrane-bending shell, 1a would need to use regular interactions to assemble a connected matrix such as the hexagonal arrays induced by the capping domain [24]. The results with BMV parallel the HIV-1 capsid protein, which contains separately interacting regions that form stacked hexamer rings www.sciencedirect.com
and is thought to induce a protein lattice that causes the membrane to curve [26]. As seen in immature HIV-1 virions, curvature of the membrane-associated lattice might be accommodated in whole or in part by gap discontinuities [26].
Host genes involved in spherule formation Genetic and genomic screens have identified >100 genes whose loss inhibits or enhances BMV RNA replication [8,27,28]. The following two sections will cover recent indepth analysis showing that host factors that regulate proper membrane composition and fluidity or affect the generation and/or maintenance of virus-induced membrane rearrangements are essential for proper spherule formation and function.
Host genes that regulate lipid metabolism and membrane composition
Lipids are the major components of cellular membranes and thus, play crucial roles in the replication of BMV and other (+) RNA viruses [29]. A mutation in OLE1-encoded D9 fatty acid desaturase, which converts saturated fatty acid to unsaturated FA (UFA), reduces BMV RNA replication by 20-fold even though it only reduces UFA levels by 12% and has no effect on cell growth or morphology [30]. Electron microscopic analysis suggests that the decrease in UFA levels preferentially affects the lipid composition of the membranes surrounding the spherules compared to the rest of the perinuclear ER membrane (Figure 2d) [31]. Multiple genes related to OLE1 expression, such as DOA4, BRO1, SPT23, and MGA2 are also involved in BMV replication in yeast [32] (Figure 1). High levels of UFAs are also required for the replication of another Bromoviridae family member, Cucumber mosaic virus [33]. These results suggest that viral replication is highly sensitive to altered lipid composition, presenting a promising opportunity to control replication of positivestrand RNA viruses. ACB1-encoded acyl-CoA binding protein (ACBP) specifically binds long-chain fatty acyl-CoAs, is highly conserved among all eukaryotes, and plays an important role in maintaining lipid homeostasis [34]. Deleting ACB1 reduces BMV replication by up to 30-fold and results in spherules that are 2-fold smaller in diameter but 4-fold more abundant than those in wt cells [35] (Figure 2e). The defects in BMV RNA replication and spherule size can be largely complemented by supplementing lipids into the growth media, suggesting that altered lipid composition is the major factor responsible for the inhibition in BMV replication [35]. The defective BMV replication phenotypes in cells lacking ACB1 phenocopy those of 1a Class II mutants (Figure 2b,e), confirming that the intimate interplay between 1a Helix A and host membranes is essential for proper replication complex assembly. Current Opinion in Virology 2014, 9:104–110
108 Special Section: Roseoloviruses
Host genes that play key roles in inducing/stabilizing membrane curvature
The reticulons are a group of ER membrane-shaping proteins that partition to and stabilize highly curved ER membrane tubules [36]. The ability of the reticulons to localize to and form tubular ER domains depends on their oligomerization, suggesting that scaffolding might play a role in curvature induction and stabilization [37]. Consistent with their function, the reticulons primarily localize to peripheral ER tubules while avoiding the lowcurvature domains of the nuclear envelope. The reticulons interact with BMV 1a and, through this interaction, relocalize from peripheral ER tubules into the interior of the replication compartments [4]. Interestingly, progressively deleting the reticulons first modulates and then abolishes spherule formation and BMV RNA replication [4]. Topologically, the body of a spherule has negative curvature as the membrane is curved away from the cytoplasm while the neck, which can be thought of as a halftubule, has positive curvature. As the reticulons are involved in forming nuclear pores [38], which are topologically equivalent to spherule necks, they are probably necessary for both spherule formation and maintenance of the neck (Figure 1). As partial reticulon depletion results in spherules of smaller diameter [24] (Figure 2f), in this instance the reticuloninduced positive-curvature might partially cancel the negative curvature of adjacent 1a proteins, causing the membrane to bend toward closure more slowly and increasing spherule diameter relative to a pure 1a shell (Figure 1). Thus, in addition to 1a, the reticulons also play key roles in spherule formation and function.
Membrane remodeling in plants infected by Bromoviruses In BMV-infected barley protoplasts, 1a, 2apol, and newly synthesized RNAs show a nearly complete co-localization with ER markers and are found adjacent to or surrounding the nucleus [19]. Various ER-derived vesicular structures have been documented in natural plant infections by BMV and its close relatives Cowpea chlorotic mosaic virus and Broad bean mosaic virus [39,40]. Similar invaginations in other membranes are present in plants infected by other Bromoviridae [41], Tombusviridae [42], and Tymoviridae [43], and in animal cells infected by alphaviruses [44–46]. However, BMV-infected Nicotiana benthamiana showed the presence of three types of cytoplasmic ERderived polymorphic vesicles, none of which localize to the perinuclear ER region as observed in yeast [47]. Type 1 vesicles are 66 nm in diameter and contain fibrillar material, with a few spherule-like structures that remain connected to the ER through neck-like structures and resemble those formed in yeast. Type 2 vesicles are 359 nm in diameter and appear to be induced by the capsid protein, while type 3 vesicles are speculated to form as a result of a fusion between type 1 and type 2 vesicles [47]. Current Opinion in Virology 2014, 9:104–110
It is probably that the differences in BMV-induced membrane rearrangements are due to the host systems used, the organization and lipid composition of the ER in yeast versus plants or possible variation in relative protein expression levels, and replication efficiency in each system. Along these lines, the membrane ultrastructure of functional BMV replication compartments can be modulated dramatically by mutating or changing the expression levels of multiple viral or cellular components, as is the case when 2apol is overexpressed or smaller spherules are formed when a single reticulon member is lacking [4,21].
Conclusions and future perspectives The work summarized herein has led to a working model where multiple 1a–1a, 1a–membrane, and 1a–host factor interactions mediate the invagination of the ER membrane as well as regulate spherule size (Figure 1). Nevertheless, new questions about our understanding of the membrane deformation process provide opportunities for further research and development. Obtaining a 1a crystal structure may provide molecular details of how 1a selfinteracts and may reveal new functional domains of 1a that are not predicted from the primary sequence. CryoEM tomography and superresolution imaging should shed further light into the detailed ultrastructure of BMV-induced membrane rearrangements as well as the organization at molecular resolution of BMV and host proteins that are part of the replication compartments. Further understanding how altered lipid composition affects 1a–1a and 1a–lipid interactions or the relocalization or activity of host proteins required for membrane remodeling may inform us spherule size and numbers are regulated. Moreover, potential interactions of 1a with other cellular pathways contributing to virus replication, such as factors that regulate membrane synthesis/composition, trafficking, and membrane remodeling remains to be explored. Additionally, determining the liposome and proteome of purified replication compartments should help identify in more detail the viral and cellular components of these compartments. The insights obtained from BMV appear relevant to many other positive-strand RNA viruses and suggest new targets for therapeutic intervention of RNA virus replication.
Acknowledgements We would like to thank Dr. Paul Ahlquist for critical discussions on the review subjects. Research in AD laboratory is supported by a startup fund from La Sierra University. Research in XW lab is supported by NSF grant IOS1265260 and by funds from Virginia Tech University. We apologize to all colleagues whose work could not be cited due to space limitations.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest www.sciencedirect.com
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ScienceDirect Bromovirus-induced remodeling of host membranes during viral RNA replication Arturo Diaz1 and Xiaofeng Wang2 With its high yield, small genome, and ability to replicate in the yeast Saccharomyces cerevisiae, Brome mosaic virus (BMV) has served as a productive model to study the general features of positive-strand RNA virus infection. BMV RNA is replicated in spherules, vesicle-like invaginations of the outer perinuclear endoplasmic reticulum membrane that remain connected to the cytoplasm via a neck-like opening. Each spherule contains the viral replicase proteins as well as genomic RNAs. Recent advances indicate that multiple interactions between the viral proteins with themselves, cellular membranes, and host factors play crucial roles in BMV-mediated spherule formation. These findings are probably applicable to other positive-strand RNA viruses and might potentially provide new targets for antiviral treatments. Addresses 1 Department of Biology, La Sierra University, Riverside, CA 92505, United States 2 Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech University, Blacksburg, VA 24061, United States Corresponding authors: Diaz, Arturo ([email protected]) and Wang, Xiaofeng ([email protected])
Current Opinion in Virology 2014, 9:104–110 This review comes from a themed issue on Special Section: Roseoloviruses Edited by Laurie Krug
http://dx.doi.org/10.1016/j.coviro.2014.09.018 1879-6257/Published by Elsevier B.V.
life cycle. Because of the limited coding capacity of positive-strand RNA viruses, the formation and function of the viral replication compartments require complex interactions between the viral proteins, viral genome, and co-opted host factors. Recent work has shown that cellular host proteins that either structurally induce and/or support membrane curvature are required to form the replication compartments [2,3,4]. Moreover, cellular lipid synthesis and appropriate lipid composition are essential for viral replication [5], implying that the membrane is an essential, functional component of the RNA replication compartments. Host membrane remodeling and the role of host genes in viral replication of other positive-strand RNA viruses have been summarized in the following reviews [1,3,6,7]. This review will focus on recent advances on the formation of the BMV-induced RNA replication complexes. Brome mosaic virus (BMV) is the type member of the Bromoviridae family and a representative member of the alphavirus-like superfamily of human, animal, and plant positive-strand RNA viruses. With its high yield, tripartite genome and ability to replicate in the yeast Saccharomyces cerevisiae, BMV has served as a productive model to study some of the general features of positive-strand RNA virus infection, including viral genomic RNA replication, gene expression, recombination, and virus–host interactions [8]. In the following sections we will summarize recent work that has enhanced our understanding of BMV replication complex assembly and structure, both of which are probably applicable to other positive-strand RNA viruses as seemingly diverse membrane rearrangements may represent topologically and functionally related structures.
General features of spherule formation Introduction The RNA replication compartments of positive-strand RNA viruses are mini-organelles that feature the close association of both viral proteins and host cellular components [1]. Viral RNA replication takes place in close association with cellular membranes and although the architecture of these compartments differs among viral families, they usually involve membrane vesicle formation or other membrane rearrangements. Cellular membranes provide a scaffold for the enrichment of the viral replication factors as well as exploited cellular proteins, provide an environment to protect the viral RNA from cellular defense mechanisms and help to separate and coordinate the various stages of the viral Current Opinion in Virology 2014, 9:104–110
BMV has three genomic RNAs and one subgenomic mRNA. RNA1 encodes the multifunctional replication factor 1a, which contains an N-proximal RNA capping domain that is separated from a C-terminal NTPase/RNA helicase-like domain by a short proline-rich sequence that may be a flexible spacer [9–12]. 2apol is encoded from RNA2 and contains a central polymerase domain and an N-terminal domain that interacts with the helicase-like domain of 1a [13,14]. Genomic RNA3 encodes the 3a movement protein, which is required for cell-to-cell spread in plants, while subgenomic RNA4 encodes for the coat protein [15]. In both yeast and plant cells, BMV RNA replication depends on the viral 1a and 2apol proteins and specific www.sciencedirect.com
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cis-acting RNA signals [16], generates a considerable excess of positive-strand to negative-strand RNA [17], and efficiently directs subgenomic RNA4 synthesis [17]. In yeast, in the absence of other viral factors, 1a induces 60–80 nm vesicular invaginations in the outer membrane of the perinuclear endoplasmic reticulum (ER), which are referred to as spherules [18] (Figure 1). Spherules are bounded by a single lipid bilayer and the interior of these vesicles is connected to the cytoplasm through a neck-like opening, probably providing a channel for ribonucleotide import and product RNA export [18]. Confocal microscopy shows that 1a accumulates in discrete ER patches that expand during the course of infection [19] while biochemical and immunogold labeling analyses suggest that there are hundreds of 1a’s per spherule and only a few copies of 2apol [18]. Consistent with this, 1a interacts with and recruits 2apol to the perinuclear ER [18,19,20]. The 1a-mediated recruitment and stabilization of genomic RNA appear to include two steps, a 1a-induced recruitment of RNA to the ER membrane followed by the translocation of the RNA into preformed spherules [12]. Moreover, incorporation of BrUTP shows nascent RNA synthesis occurs within the spherules [18] (Figure 1). However, modulating the relative levels and interactions between replication factors 1a and 2apol shifts the membrane rearrangements from spherular compartments to large and multilayer stacks of appressed double-membrane
layers that support RNA replication as efficiently as spherules [21]. The following two sections will summarize recent work that sheds light into the manner by which 1a interacts with itself, lipid membranes, and other viral components and the details by which it invaginates the ER membranes to induce spherule formation. Roles of Helix A
1a has no trans-membrane domain and fully resides on the cytoplasmic side of the ER membrane [22]. A small amphipathic a-helix, termed Helix A, is crucial for both 1a’s membrane association and 1a-induced membrane rearrangement as well as 1a-mediated recruitment of viral RNA templates and 2apol [22,23] (Figure 1). Genetic, biochemical, and NMR analyses showed that mutations within Helix A give rise to two classes of mutants with distinct properties. Class I mutants, which disrupt helix formation, significantly inhibit 1a membrane affinity and fail to induce spherule formation but are over twice as effective as wild-type (wt) 1a at stabilizing and recruiting 2apol to nonperinuclear ER membranes [23]. By contrast, Class II mutants interact with 2apol inefficiently but increase the frequency of 1a-induced spherules by 5-fold, although these compartments are 30% smaller in diameter than those induced by wt 1a [23] (Figure 2b). Correspondingly, Class I mutants fail to recruit the viral
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Interactions and host factors required for the formation of functional BMV RNA replication complexes. 1a and 2apol interact in the cytoplasm before membrane association. 1a associates with the outer ER membrane, where 1a–1a and 1a–membrane interactions, in conjunction with host factors, lead to the invagination of the ER membrane to form spherules. 1a-mediated recruitment of viral RNA templates occurs after the formation of spherules, followed by the synthesis and retention of negative-strand RNA (dashed black lines), and asymmetric synthesis and export of positive-strand progeny RNA (red lines). Host factors involved in maintaining proper membrane lipid composition around the spherule membranes and in generating and/or maintaining the virus-induced membrane rearrangements are shown. www.sciencedirect.com
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Figure 2
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BMV-induced membrane rearrangements formed under various conditions. Representative electron micrographs of spherules made by (a) wt 1a or (b) 1a with a Class II mutation. (c) Hexagonal arrays induced by the N-terminal capping domain of 1a. Spherules made in (d) ole1w, (e) acb1D, or (f) rtn1D mutant yeast strains. Arrows point out individual spherules. Nuc: nucleus; Cyto: cytoplasm. Scale bars, 200 nm. Electron micrographs are reproduced with permission from [4,23,24,30,35] and [4], respectively.
RNA while Class II mutants enhance RNA recruitment to even higher levels than wt 1a, further showing that RNA recruitment and protection by 1a strongly correlate with 1ainduced spherule formation [23]. Current Opinion in Virology 2014, 9:104–110
These results imply that the 1a–2apol interactions occur before spherule formation and that 1a–2apol interaction and 1a-induced spherule formation are sequential and perhaps antagonistic [23] (Figure 1). Moreover, the fact www.sciencedirect.com
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that Class II mutants alter spherule sizes suggests that 1a– membrane and/or 1a–1a interactions influence the diameter of these structures. 1a–1a interactions and multimerization
To further identify the determinants of spherule formation, the N-terminal capping and C-terminal helicaselike domains of BMV 1a were separately examined. Although both the capping and helicase domains individually multimerize, neither domain on its own induces spherule formation [24]. In vivo, the 1a capping domain forms membrane-linked hexagonal lattices of tubules or stacked rings and induces layering of ER double membranes while the helicase domain fails to associate with membranes [24] (Figure 2c). When expressed in trans, the capping domain fails to recruit the helicase domain to ER membranes, consistent with prior yeast two-hybrid results implying that the capping and helicase domains interact intramolecularly within full-length 1a but do not significantly interact intermolecularly between two 1a’s [25]. Moreover, when co-expressed, the capping domain recruits full-length 1a into hexagonal tubule lattices and inhibits 1a’s ability to form spherules and support RNA replication [24]. The results suggest at least three different interactions between 1a proteins: intramolecular interactions between the capping and helicase-like domains, intermolecular interactions between capping domains, and independent intermolecular interactions between helicase-like domains (Figure 1). It is possible that all of these contacts form simultaneously such that 1a molecules contact one another at multiple interfaces or that some interactions or multimeric states may be specific to certain 1a functions. The sum of the intramolecular and inter-molecular interactions appears to lead not only to the emergence of an extensive multimeric 1a lattice but also to the invagination of the perinuclear ER membrane that results in the generation of spherules. Disrupting any of those interactions inhibits spherule formation, for example, 1a-induced membrane rearrangements shift from spherules to appressed double-membrane layers when the helicase interacting 2apol is overexpressed [21], probably by interfering with helicase–helicase interactions between adjacent 1a proteins. 1a’s strong membrane association and asymmetric insertion of Helix A into the lipid bilayer to generate local membrane curvature [22,23], its ability to self-interact [24,25], and its high multiplicity within spherules [18] suggest that 1a forms a shell lining to induce and maintain the vesicular replication compartments (Figure 1). In order to form such a membrane-bending shell, 1a would need to use regular interactions to assemble a connected matrix such as the hexagonal arrays induced by the capping domain [24]. The results with BMV parallel the HIV-1 capsid protein, which contains separately interacting regions that form stacked hexamer rings www.sciencedirect.com
and is thought to induce a protein lattice that causes the membrane to curve [26]. As seen in immature HIV-1 virions, curvature of the membrane-associated lattice might be accommodated in whole or in part by gap discontinuities [26].
Host genes involved in spherule formation Genetic and genomic screens have identified >100 genes whose loss inhibits or enhances BMV RNA replication [8,27,28]. The following two sections will cover recent indepth analysis showing that host factors that regulate proper membrane composition and fluidity or affect the generation and/or maintenance of virus-induced membrane rearrangements are essential for proper spherule formation and function.
Host genes that regulate lipid metabolism and membrane composition
Lipids are the major components of cellular membranes and thus, play crucial roles in the replication of BMV and other (+) RNA viruses [29]. A mutation in OLE1-encoded D9 fatty acid desaturase, which converts saturated fatty acid to unsaturated FA (UFA), reduces BMV RNA replication by 20-fold even though it only reduces UFA levels by 12% and has no effect on cell growth or morphology [30]. Electron microscopic analysis suggests that the decrease in UFA levels preferentially affects the lipid composition of the membranes surrounding the spherules compared to the rest of the perinuclear ER membrane (Figure 2d) [31]. Multiple genes related to OLE1 expression, such as DOA4, BRO1, SPT23, and MGA2 are also involved in BMV replication in yeast [32] (Figure 1). High levels of UFAs are also required for the replication of another Bromoviridae family member, Cucumber mosaic virus [33]. These results suggest that viral replication is highly sensitive to altered lipid composition, presenting a promising opportunity to control replication of positivestrand RNA viruses. ACB1-encoded acyl-CoA binding protein (ACBP) specifically binds long-chain fatty acyl-CoAs, is highly conserved among all eukaryotes, and plays an important role in maintaining lipid homeostasis [34]. Deleting ACB1 reduces BMV replication by up to 30-fold and results in spherules that are 2-fold smaller in diameter but 4-fold more abundant than those in wt cells [35] (Figure 2e). The defects in BMV RNA replication and spherule size can be largely complemented by supplementing lipids into the growth media, suggesting that altered lipid composition is the major factor responsible for the inhibition in BMV replication [35]. The defective BMV replication phenotypes in cells lacking ACB1 phenocopy those of 1a Class II mutants (Figure 2b,e), confirming that the intimate interplay between 1a Helix A and host membranes is essential for proper replication complex assembly. Current Opinion in Virology 2014, 9:104–110
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Host genes that play key roles in inducing/stabilizing membrane curvature
The reticulons are a group of ER membrane-shaping proteins that partition to and stabilize highly curved ER membrane tubules [36]. The ability of the reticulons to localize to and form tubular ER domains depends on their oligomerization, suggesting that scaffolding might play a role in curvature induction and stabilization [37]. Consistent with their function, the reticulons primarily localize to peripheral ER tubules while avoiding the lowcurvature domains of the nuclear envelope. The reticulons interact with BMV 1a and, through this interaction, relocalize from peripheral ER tubules into the interior of the replication compartments [4]. Interestingly, progressively deleting the reticulons first modulates and then abolishes spherule formation and BMV RNA replication [4]. Topologically, the body of a spherule has negative curvature as the membrane is curved away from the cytoplasm while the neck, which can be thought of as a halftubule, has positive curvature. As the reticulons are involved in forming nuclear pores [38], which are topologically equivalent to spherule necks, they are probably necessary for both spherule formation and maintenance of the neck (Figure 1). As partial reticulon depletion results in spherules of smaller diameter [24] (Figure 2f), in this instance the reticuloninduced positive-curvature might partially cancel the negative curvature of adjacent 1a proteins, causing the membrane to bend toward closure more slowly and increasing spherule diameter relative to a pure 1a shell (Figure 1). Thus, in addition to 1a, the reticulons also play key roles in spherule formation and function.
Membrane remodeling in plants infected by Bromoviruses In BMV-infected barley protoplasts, 1a, 2apol, and newly synthesized RNAs show a nearly complete co-localization with ER markers and are found adjacent to or surrounding the nucleus [19]. Various ER-derived vesicular structures have been documented in natural plant infections by BMV and its close relatives Cowpea chlorotic mosaic virus and Broad bean mosaic virus [39,40]. Similar invaginations in other membranes are present in plants infected by other Bromoviridae [41], Tombusviridae [42], and Tymoviridae [43], and in animal cells infected by alphaviruses [44–46]. However, BMV-infected Nicotiana benthamiana showed the presence of three types of cytoplasmic ERderived polymorphic vesicles, none of which localize to the perinuclear ER region as observed in yeast [47]. Type 1 vesicles are 66 nm in diameter and contain fibrillar material, with a few spherule-like structures that remain connected to the ER through neck-like structures and resemble those formed in yeast. Type 2 vesicles are 359 nm in diameter and appear to be induced by the capsid protein, while type 3 vesicles are speculated to form as a result of a fusion between type 1 and type 2 vesicles [47]. Current Opinion in Virology 2014, 9:104–110
It is probably that the differences in BMV-induced membrane rearrangements are due to the host systems used, the organization and lipid composition of the ER in yeast versus plants or possible variation in relative protein expression levels, and replication efficiency in each system. Along these lines, the membrane ultrastructure of functional BMV replication compartments can be modulated dramatically by mutating or changing the expression levels of multiple viral or cellular components, as is the case when 2apol is overexpressed or smaller spherules are formed when a single reticulon member is lacking [4,21].
Conclusions and future perspectives The work summarized herein has led to a working model where multiple 1a–1a, 1a–membrane, and 1a–host factor interactions mediate the invagination of the ER membrane as well as regulate spherule size (Figure 1). Nevertheless, new questions about our understanding of the membrane deformation process provide opportunities for further research and development. Obtaining a 1a crystal structure may provide molecular details of how 1a selfinteracts and may reveal new functional domains of 1a that are not predicted from the primary sequence. CryoEM tomography and superresolution imaging should shed further light into the detailed ultrastructure of BMV-induced membrane rearrangements as well as the organization at molecular resolution of BMV and host proteins that are part of the replication compartments. Further understanding how altered lipid composition affects 1a–1a and 1a–lipid interactions or the relocalization or activity of host proteins required for membrane remodeling may inform us spherule size and numbers are regulated. Moreover, potential interactions of 1a with other cellular pathways contributing to virus replication, such as factors that regulate membrane synthesis/composition, trafficking, and membrane remodeling remains to be explored. Additionally, determining the liposome and proteome of purified replication compartments should help identify in more detail the viral and cellular components of these compartments. The insights obtained from BMV appear relevant to many other positive-strand RNA viruses and suggest new targets for therapeutic intervention of RNA virus replication.
Acknowledgements We would like to thank Dr. Paul Ahlquist for critical discussions on the review subjects. Research in AD laboratory is supported by a startup fund from La Sierra University. Research in XW lab is supported by NSF grant IOS1265260 and by funds from Virginia Tech University. We apologize to all colleagues whose work could not be cited due to space limitations.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest www.sciencedirect.com
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