Poliovirus RNA is released from the capsid near a twofold symmetry axis

After recognizing and binding to its host cell, poliovirus (like other nonenveloped viruses) faces the challenge of translocating its genome across a cellular membrane and into the cytoplasm. To avoid entanglement with the capsid, the RNA must exit via a single site on the virion surface. However, the mechanism by which a single site is selected (from among 60 equivalents) is unknown; and until now, even its location on the virion surface has been controversial. To help to elucidate the mechanism of infection, we have used single-particle cryo-electron microscopy and tomography to reconstruct conformationally altered intermediates that are formed by the poliovirion at various stages of the poliovirus infection process. Recently, we reported icosahedrally symmetric structures for two forms of the end-state 80S empty capsid particle. Surprisingly, RNA was frequently visible near the capsid; and in a subset of the virions, RNA was seen on both the inside and outside of the capsid, caught in the act of exiting. To visualize RNA exiting, we have now determined asymmetric reconstructions from that subset, using both single-particle cryo-electron microscopy and cryo-electron tomographic methods, producing independent reconstructions at ∼50-Å resolution. Contrary to predictions in the literature, the footprint of RNA on the capsid surface is located close to a viral 2-fold axis, covering a slot-shaped area of reduced density that is present in both of the symmetrized 80S reconstructions and which extends by about 20 Å away from the 2-fold axis toward each neighboring 5-fold axis.In its role as the intermediate that links one round of infection with the next, a virus particle protects the viral genome during passage from cell to cell and from host to host, it specifically recognizes and binds to target cells, and it delivers the viral genome into the appropriate compartment in the target cell. For enveloped viruses, which have their own external membranes, fusion of the viral membrane with a host membrane presents a conceptually simple mechanism for delivery of the genome or nucleoprotein into the cytoplasm. For nonenveloped viruses, the viral particle must provide the machinery necessary for either the entire virion, a nucleoprotein complex, or the viral genome to cross a membrane. This process remains poorly understood. Poliovirus provides an excellent model system for probing the mechanisms used for genome translocation. As the type member of the Picornavirus family and the etiological agent of poliomyelitis, poliovirus has been well characterized biochemically and genetically (42), its cell entry pathways have been well characterized (5, 15, 30, 52), and a number of cell entry intermediates have been identified and are accessible for structural studies (2-4, 7, 8, 18, 34, 38, 42, 55, 56).The capsid of the mature poliovirion (160S particle) consists of 60 copies of each of the four coat proteins VP1, VP2, VP3, and VP4 (which is myristolated at its amino-terminal glycine [13]) and encloses a 7.5-kbp positive-sense RNA genome. The outer surface of the capsid has a number of major features, including star-shaped mesas at its 5-fold axes, 3-fold propeller-like protrusions, canyon-like depressions surrounding each of the 5-fold mesas, and depressions at the 2-fold axes (30, 31).Poliovirus infection is initiated when the virus binds to the host-cell-surface poliovirus receptor (called Pvr or CD155) (41), triggering a conformational change of the native capsid into an altered particle called the A particle or 135S particle (18, 19). The 135S particle has been shown to be expanded by about 4% (2, 7), is infectious (16, 33), and is believed to be a productive intermediate in viral entry (30, 33). This conformational change results in the externalization of the small myristoylated capsid protein, VP4 (18), and of the amino-terminal extension of VP1 (which includes a conserved amphipathic helix) (23). Both of these externalized polypeptides then associate with membranes (17, 23). In subsequent steps, the viral genome is released from the capsid and translocated across a membrane (probably an endosomal membrane [5]) to gain access to the cytoplasm, leaving behind an end-state empty capsid shell (called the 80S particle). The trigger for RNA release and the mechanism of genome translocation are both poorly understood (30, 52).Electrophysiology and mutational experiments have shown that the externalization of VP4 and of the amino terminus of VP1 is associated with the formation of channels in membranes (17, 49, 50) and, furthermore, that point mutations in threonine 28 of VP4 can either eliminate (T28G) or alter (T28V, T28S) the ability to form channels and either eliminate (T28G) or slow (T28V, T28S) the kinetics of productive RNA release (17). These observations have led to the hypothesis that the viral polypeptides insert into host cell membranes during infection and rearrange to form channels that permit the viral genome to pass through the membrane, thereby gaining access to the cytoplasm (7, 17, 49, 50).Speculation about the sites of externalization of the viral peptides and of the viral genome began soon after the structures of mature rhinovirus and poliovirus were determined crystallographically 25 years ago (31, 44). In both structures there is a solvent-filled channel running along each 5-fold axis. This channel is closed off at the outer surface of the capsid by polypeptide loops and on the inner surface by a plug that is formed by five intertwined copies of the amino terminus of VP3, forming a parallel beta tube (31, 44). In poliovirus this tube is flanked on its inner surface by five copies of a three-stranded beta sheet in which the outermost two strands come from a beta hairpin at the amino terminus of VP4 and the innermost strand comes from residues at the extreme amino terminus of VP1 (20). The presence of this channel, together with its proximity to peptide segments that were known to be externalized upon receptor attachment, and analogies with other viruses led to a model in which both the peptides and the viral RNA are externalized via the channel at the 5-fold axis (25, 45). At that time, an alternative model for the egress of polypeptides was proposed, based on an analogy with the externalization of the amino-terminal extensions of capsid proteins in expanded states of the topologically similar T=3 plant viruses (26, 32, 43, 47) and on genetic and biochemical studies of mutations that affect cell entry and capsid stability in poliovirus (14, 39, 54). In the latter model, the peptides were proposed to exit from the base of the canyon and then proceed along the outer surface toward the 5-fold peak (43, 47). Both models suggested that five copies of each of the externalized peptides would interact in some way to form a pore in the membrane that was contiguous with one of the 5-fold channels, thus providing a way for RNA to be released from the virion at a 5-fold axis of symmetry. No data yet exist to specify what specific structural roles VP4 and the amino terminus of VP1 might play in forming pores and serving as membrane anchors. However, both the electrophysiology data (cited above) and the greater sequence conservation of VP4 suggest that its role in pore formation may be the more central (17, 49, 50).To further elucidate various steps along the infection pathway, cryo-electron microscopy (cryo-EM) reconstructions have been determined for a number of cell entry intermediates of poliovirus and rhinoviruses, and their resolutions have been improved over time (2, 3, 7, 28, 38). Structures of the complexes of polioviruses and major-group rhinoviruses with the ectodomains of their respective receptors have confirmed earlier models that suggested that the canyon is the receptor-binding site and have begun to suggest how receptor binding might lead to receptor-induced conformational rearrangements (3, 56). Cryo-EM and cryo-electron tomography structures (cryo-ET) of a poliovirus-receptor-membrane complex (using a novel receptor-decorated liposome model [51]) confirmed that initial receptor binding brings the surface of the 5-fold mesa into close proximity with the membrane and appears to produce an outward distortion of the outer leaflet of the membrane in its area of closest approach to the virus particle (4, 8).Structures have also been determined for the soluble 135S and 80S particles of poliovirus, formed by heating the virus at 50°C (135S) or 56°C (80S) in hypotonic buffers, and for the 80S particles of rhinovirus 14 and 16, formed by exposing virus to acidic pH. All of the biological and immunological evidence that is currently available indicates that the particles prepared in vitro and used for structural studies are indistinguishable from the particles that are released from the cell surface during infection (6, 53). These structures have allowed the models for peptide release and genome release to be extended and refined (7, 38) and indeed have confirmed that VP1 exits from the particle surface at the base of the canyon and climbs up the side of the 5-fold mesa. However, contrary to the assumptions of the earlier models, the 10-Å structures of the poliovirus 135S and 80S particles show that the amino end of the amino-terminal extension of VP1 does not remain associated with the mesa. Instead, it forms an alpha-helical bridge that stretches across the canyon and binds to the large EF loop of VP2, a surface projection that appears as a 3-fold propeller blade (7, 38).Until recently, the mechanism of RNA release (during the 135S-to-80S transition) has been largely a matter of conjecture. We can infer that the RNA must exit via a single site on the virion surface, to avoid entanglement with the capsid (particularly as entanglement has never been observed in electron micrographs), though the mechanism by which a single site is selected (from among 60 equivalents) is unknown. All models presented to date have assumed that the RNA is released from the channel at the 5-fold axes (2, 3, 7, 8, 25, 27, 28, 30, 42, 45). However, in the icosahedrally constrained 10-Å structures of both the poliovirus 135S and 80S particles (7, 38), the apparent intactness and stability of the 5-fold mesa argues against the 5-fold axis being the site of RNA egress, given that the diameter of the opening, as seen in those structures, would be insufficient to accommodate RNA, even if the “plug” formed by the intertwined amino termini of VP3 was displaced. Moreover, both structures revealed significant thinning between 2-fold-related pentamers in the vicinity of the 2-fold axes. Most convincingly, large holes (easily sufficient to accommodate RNA) were seen at and near the 2-fold axes in the atomic model of the late-80S structure. This coincided with an open hole in the reconstruction, when viewed at a contour level that left most of the remainder of the capsid intact. This evidence was suggestive, but not definitive, as a number of other openings were present, particularly in the interfaces between protomers. Furthermore, the behavior of the capsid structure in the immediate vicinity of the unique site of RNA exit is likely to be different from what we see in the icosahedral average, which is dominated by the remainder of the capsid.In the course of solving icosahedrally symmetric cryo-EM structures for the poliovirus end-state 80S empty capsid particle (7, 38), we were surprised to find that RNA was frequently visible near the capsid and that in a subset of about 5% of the sampled virions, RNA was seen on both the inside and outside of the capsid, apparently caught in the act of exiting. This was an exciting development, as images of viral RNA release had never previously been reported. We were able to improve the resolution to ∼10 Å by classifying the projected images into two groups: an early 80Se particle that was more prevalent in the population after a shorter heating time and a late 80Sl particle that was seen more often when the heating time was increased. The amount of RNA density remaining in the interior appears to be continuously variable in both classes, suggesting that release is gradual. Of the 5% subset of particles clearly caught in the act, almost all belonged to the 80Se class. Our interpretation was that the 80Se class may represent particles in which exiting RNA is still engaged with the capsid machinery and traversing the capsid, while the 80Sl class (in which much of the capsid resembles the 135S form more closely in structure) represents particles with the RNA disengaged, possibly after nuclease cleavage. More than two structural classes may be present, but at the current resolution, we could not distinguish them.The present report addresses the question of what we can learn about the details of RNA release from an asymmetric cryo-EM reconstruction, based on the 540-particle caught-in-the-act subset, and independently from cryo-electron tomographic reconstructions of a similarly prepared sample. In each projected particle image or subtomogram, preliminary orientation parameters are first determined from an icosahedrally symmetric calculation, and in a second stage, the symmetry is broken by choosing 1 of the 60 symmetry-equivalent orientations. Both methods have yielded similar information, at about 50-Å resolution, concerning the footprint of the RNA on the virion surface, which demonstrates that RNA is released from an asymmetric site at the base of the canyon near a particle 2-fold axis and not at the channel at the 5-fold axes, as suggested by previous models. Additionally, the demonstrated success of the methodology provides us with a blueprint for resolving the molecular details of the RNA-capsid interaction in future experiments.     

A Targeted Analysis of Cellular Chaperones Reveals Contrasting Roles for Heat Shock Protein 70 in Flock House Virus RNA Replication

Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.The compact genomes of viruses relative to those of other infectious agents restrict their ability to encode all proteins required to complete their replication cycles. To circumvent this limitation, viruses often utilize cellular factors or processes to complete essential steps in replication. One group of cellular proteins frequently targeted by viruses are cellular chaperones, which include a diverse set of heat shock proteins (hsps) that normally facilitate cellular protein translation, folding, trafficking, and degradation (18, 64). The connection between viruses and cellular chaperones was originally identified in bacteria, where the Escherichia coli hsp40 and hsp70 homologues, encoded by dnaJ and dnaK, respectively, were identified as bacterial genes essential for bacteriophage λ DNA replication (62). Research over the past 30 years has further revealed the importance of cellular chaperones in viral replication, such that the list of virus-hsp connections is now quite extensive and includes viruses from numerous families with diverse genome structures (4, 6, 7, 16, 19, 20, 23, 25, 40, 41, 44, 51, 54, 60). These studies have demonstrated the importance of cellular chaperones in multiple steps of the viral life cycle, including entry, viral protein translation, genome replication, encapsidation, and virion release. However, the list of virus-hsp connections is likely incomplete. Further studies to explore this particular host-pathogen interaction will shed light on virus replication mechanisms and pathogenesis, and potentially highlight targets for novel antiviral agents.To study the role of cellular chaperones in the genome replication of positive-sense RNA viruses, we use flock house virus (FHV), a natural insect pathogen and well-studied member of the Nodaviridae family. The FHV life cycle shares many common features with other positive-sense RNA viruses, including the membrane-specific targeting and assembly of functional RNA replication complexes (37, 38), the exploitation of various cellular processes and host factors for viral replication (5, 23, 60), and the induction of large-scale membrane rearrangements (24, 28, 38, 39). FHV virions contain a copackaged bipartite genome consisting of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode protein A, the viral RNA-dependent RNA polymerase, and the structural capsid protein precursor, respectively (1). During active genome replication, FHV produces a subgenomic RNA3 (0.4 kb), which encodes the RNA interference inhibitor protein B2 (12, 29, 32). These viral characteristics make FHV an excellent model system to study many aspects of positive-sense RNA virus biology.In addition to the benefits of a simple genome, FHV is able to establish robust RNA replication in a wide variety of genetically tractable eukaryotic hosts, including Drosophila melanogaster (38), Caenorhabditis elegans (32), and Saccharomyces cerevisiae (46). The budding yeast S. cerevisiae has been an exceptionally useful model host to study the mechanisms of viral RNA replication complex assembly and function with FHV (31, 37, 39, 45, 53, 55, 56, 60) as well as other positive-sense RNA viruses (11). The facile genetics of S. cerevisiae, along with the vast array of well-defined cellular and molecular tools and techniques, make it an ideal eukaryotic host for the identification of cellular factors required for positive-sense RNA virus replication. Furthermore, readily available yeast libraries with deletions and regulated expression of individual proteins have led to the completion of several high-throughput screens to provide a global survey of host factors that impact virus replication (26, 42, 52). An alternative approach with these yeast libraries that reduces the inherently high false-negative rates associated with high-throughput screens is to focus on a select set of host genes associated with a particular cellular pathway, process, or location previously implicated in virus replication.We have utilized such a targeted approach and focused on examining the impact of cytosolic chaperones on FHV RNA replication. Previously, we have shown that the cellular chaperone hsp90 facilitates protein A synthesis in Drosophila cells (5, 23), and the hsp40 encoded by the yeast YDJ1 gene facilitates FHV RNA replication in yeast, in part through effects on both protein A accumulation and function (60). In this report, we further extend these observations by examining FHV RNA accumulation in a panel of yeast strains with deletions of known or hypothesized cytosolic chaperones. We demonstrate that cytosolic chaperones can have either suppressive or enhancing effects on FHV RNA accumulation. In particular, related hsp70 members encoded by the SSA and SSB yeast chaperone families have marked and dramatically divergent effects on both genomic and subgenomic RNA accumulation and viral polymerase synthesis. These results highlight the complexities of the host-pathogen interactions that influence positive-sense RNA virus replication and identify the hsp70 family of cytosolic chaperones as key regulators of FHV replication.     

A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.A novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (10, 39). SARS-CoV is an enveloped, single- and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (46, 55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensin-converting enzyme 2 (ACE2) (44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARS-CoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (46) with 23 potential N-linked glycosylation sites (55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptor-binding domain [RBD]) and S2 (containing the membrane fusion domains) (1, 31, 51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (50, 61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (46, 55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (28, 59), as well as trypsin (43, 61) and factor Xa (11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannose-binding or mannan-binding protein [MBP]) is a Ca²⁺-dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (8, 14, 18), where it activates the complement system or acts directly as an opsonin (30, 40, 52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (9) and usually has two to six clusters of CRDs. Within each of the clusters, the carbohydrate-binding sites have a fixed orientation, which allows selective recognition of patterns of carbohydrate residues on the surfaces of a wide range of microorganisms (8, 14, 18). The concentration of MBL in the serum varies greatly and is affected by mutations of the promoter and coding regions of the human MBL gene (45). MBL deficiency is associated with susceptibility to various infections, as well as autoimmune, metabolic, and cardiovascular diseases, although MBL-deficient individuals are generally healthy (13, 37, 67).There are conflicting results with regard to the role of MBL in SARS-CoV infection (29, 42, 72, 73). While the association of MBL gene polymorphisms with susceptibility to SARS-CoV infection was reported in some studies (29, 73), Yuan et al. demonstrated that there were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls (72). Ip et al. observed binding to, and inhibition of, SARS-CoV by MBL (29). However, in other studies, no binding of MBL to purified SARS-CoV S glycoprotein was detected (42).In this study, retroviral particles pseudotyped with SARS-S and in vitro assays were used to characterize the role of MBL in SARS-CoV infection. The data indicated that MBL selectively bound to SARS-S and mediated inhibition of viral infection in susceptible cell lines. Moreover, we identified a single N-linked glycosylation site, N330, on SARS-S that is critical for the specific interactions with MBL.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.