A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Lability and Confer Resistance to Acid-Dependent Uncoating Inhibition

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH₄Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH₄Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH₄Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus in the family Picornaviridae. FMDV displays epithelial tropism and is responsible for a highly contagious disease of cloven-hoofed animals (23, 60). FMDV populations are quasispecies and exhibit a high potential for variation and adaptation, one consequence of which is the extensive antigenic diversity of this virus, reflected in the existence of seven serotypes and multiple antigenic variants (reviewed in references 17 and 60). Different cellular receptors, including α_vβ integrins and heparan sulfate (HS) glycosaminoglycans, have been described for natural isolates and tissue culture-adapted FMDVs (3, 4, 6, 28-31, 56). However, viruses that are infectious in vivo use integrins as receptors (28). The interaction between FMDV and the integrin molecule is mediated by an Arg-Gly-Asp (RGD) triplet located at the G-H loop of capsid protein VP1 (9, 47). FMDV isolates interacting with integrins gain entry into the cell following clathrin-mediated endocytosis (8, 39, 52). On the other hand, it has been described that a genetically engineered HS-binding mutant uses caveolae to enter into cultured cells (51). After internalization, FMDV must release its genomic RNA molecule of positive polarity into the host cell cytoplasm to establish a productive infection. Early work showed that a variety of lysosomotropic agents, such as weak bases and ionophores that block acidification of endosomes, inhibit FMDV infection (5, 11-13), indicating that genome release is dependent on endosomal acidification. In addition, internalized FMDV particles colocalize with markers from early and recycling endosomes (8, 51, 52) and FMDV infection is reduced by expression of a dominant negative mutant of Rab5 (33), suggesting that FMDV may release its genome from these compartments.The FMDV capsid comprises 60 copies of each of the four structural proteins (VP1 to VP4) arranged in an icosahedral lattice of 12 pentameric subunits. FMDV particles are highly acid labile and disassemble at pH values slightly below neutrality (13). Acid lability is not a feature of the capsids of other picornaviruses, such as Enterovirus. Pentameric subunits are intermediates of FMDV assembly and disassembly (64). A high density of His residues is found close to the interpentameric interface. Protonation of these residues at the acidic pH in the endosomes has been proposed to trigger acid-induced capsid disassembly by electrostatic repulsion between the protonated His side chains (1). His 142 (H142) in VP3 of type A FMDV is involved in a His-α-helix dipole interaction, which is likely to influence the acid lability of FMDV (13). In silico predictions suggested that H142 and H145 in VP3 may have the greatest effect on this process (63). Experimental evidence of the involvement of H142 of VP3 in acid-induced disassembly of FMDV has also been reported (20). Concomitantly with capsid disassembly into pentameric intermediates, internal protein VP4 and viral RNA are released. VP4 is a highly hydrophobic and myristoylated protein (7) whose release has been suggested to mediate membrane permeabilization and ion channel formation, thus facilitating the endosomal exit of viral RNA (15, 16, 34).Besides providing information about the endosomal pH requirements for the release of virus genomes, drugs modifying endosomal acidification can reveal the molecular changes associated with viral resistance to their action. These analyses may also address whether the balance between acid lability and capsid stability required for completion of virus replication allows FMDV, which disassembles at a pH close to neutrality, to escape inhibition by drugs raising the endosomal pH. In this work, we have isolated and characterized FMDV mutants that are able to escape from the inhibition of endosomal acidification exerted by NH₄Cl, a lysosomotropic weak base that raises endolysosomal pH and impairs uncoating and infection of viruses that require transit through acidic endosomal compartments for penetration (5, 26, 53). These mutants showed an increased acid lability, which is likely to allow them to uncoat at more-alkaline pH values. A single amino acid substitution close to the interpentameric interfaces in the capsid of one of these mutants was responsible for a total resistance to the elevation in endosomal pH caused by NH₄Cl treatment and for the acid-labile phenotype.     

A Dominant-Negative Mutant of rab5 Inhibits Infection of Cells by Foot-and-Mouth Disease Virus: Implications for Virus Entry

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (αvβ1, αvβ3, αvβ6, and αvβ8) as receptors to initiate infection. Infection mediated by αvβ6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express αvβ8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by αvβ8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (∼35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus of the family Picornaviridae and the etiological agent responsible for FMD, an economically important and severe vesicular condition of cloven-hoofed animals, including cattle, pigs, sheep, and goats (2). The mature virus particle consists of a positive-sense single-stranded RNA genome (vRNA) enclosed within a nonenveloped icosahedral capsid formed from 60 copies each of four virus-encoded proteins, VP1 to VP4 (1).The initial stage of FMDV infection is virus binding to cell surface integrins via a highly conserved RGD motif located on the GH loop of VP1. A number of different species of RGD-binding integrins (αvβ1, αvβ3, αvβ6, and αvβ8) have been reported to serve as receptors for FMDV (5, 23-26). Using pharmacological and dominant-negative (DN) inhibitors of specific endocytic pathways in combination with immunofluorescence confocal microscopy, the cell entry pathway used by FMDV has been determined for αvβ6-expressing cells (6, 36). These studies established that infection occurs by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes, which serves as the trigger for capsid disassembly and translocation of the vRNA across the endosomal membrane into the cytosol. Internalized virus was detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomes (LE) or lysosomes (Lys) (the late endosomal compartments). Due to the extreme sensitivity of FMDV to acidic pH (15), it is thought that EE can provide a pH low enough for virus disassembly to occur; however, definitive proof that infection takes place from within EE is still lacking. For example, the possibility cannot be excluded that a productive infection requires virus transport to late endosomal compartments, where, following capsid disassembly and viral genome transfer into the cytosol, the capsid proteins are rapidly degraded.rab proteins control multiple membrane trafficking events in the cell. They are members of the ras superfamily of small GTP-binding proteins and cycle between active GTP- and inactive GDP-bound states (22, 38, 39, 47, 50). Conversion between these states is regulated by guanine nucleotide exchange factors, which stimulate the binding of GTP, and GTPase-activating proteins that which accelerate GTP hydrolysis. Activated rab proteins are recruited onto membrane-bounded compartments where they regulate many steps of vesicle trafficking, including vesicle budding, movement, tethering, and fusion (35, 61). Each rab is recruited to a specific compartment and functions through interactions with specific effectors that mediate the downstream rab-associated functions (39). In mammalian cells, at least 12 rab proteins that regulate trafficking through the endosomal pathway have been identified (27). Of these, rab4, rab5, rab7, and rab11 play major roles in endocytic vesicle trafficking. rab5 is present on EE and regulates transport of incoming endocytic vesicles from the plasma membrane (PM) to EE and homotypic EE fusion events (3, 8, 10, 20, 30, 44, 52). Both rab4 and rab11 are regulators of receptor recycling from EE back to the PM (34); rab4 is localized primarily to EE and regulates rapid recycling directly back to the PM (16, 45, 48, 51, 56), and rab11 is localized primarily to the PNRE and regulates a slower recycling pathway through these compartments (21, 43, 54, 60). In addition rab11 also regulates membrane traffic from endocytic recycling compartments to the trans-Golgi network (55). rab7 is located primarily on LE and regulates traffic from EE to LE and between LE and Lys (7, 9, 18, 32, 40, 58, 59). The unique targeting of rab proteins to distinct cellular compartments and their specificity as regulators of vesicular trafficking has made them important tools for studying endocytosis. For example, expression of DN or constitutively active mutants of rab proteins that regulate endosomal traffic has been used to identify the intracellular transport steps that are required for infection by a number of different viruses (13, 14, 28, 31, 41, 42, 49, 53, 57, 59).Here we have investigated the intracellular transport steps required for FMDV infection using porcine IBRS-2 cells, which are derived from a natural host of FMDV. IBRS-2 cells use αvβ8, and not αvβ6, as the major FMDV receptor (11). Our initial experiments confirmed that FMDV infection mediated by αvβ8 is dependent on clathrin-mediated endocytosis and on an acidic pH within endosomes. The effect on FMDV infection within IBRS-2 cells of DN mutants of cellular rab proteins that regulate endosomal traffic was examined. These experiments show that rab5 is needed for FMDV infection, as expression of DN rab5 reduced the number of FMDV-infected cells by ∼80%. In contrast, expression of either DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (∼35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.     

Cell Entry of the Aphthovirus Equine Rhinitis A Virus Is Dependent on Endosome Acidification

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.Equine rhinitis A virus (ERAV) belongs to the genus Aphthovirus of the family Picornaviridae (23) and is closely related to foot-and-mouth disease virus (FMDV) as they share physicochemical properties (18, 19), nucleotide sequence (15, 28, 34), and structural similarities (31). Picornaviruses are small nonenveloped RNA viruses, comprising a 30-nm-diameter capsid made of 60 copies of each of four capsid proteins, VP1 to VP4, which encapsidate a single-stranded RNA genome (7 to 8 kb) (24). The family has 12 genera and includes a number of important pathogens (e.g., poliovirus [PV], human rhinovirus [HRV], hepatitis A virus [HAV], etc.).Despite extensive study of FMDV, there are still many aspects of aphthovirus biology, such as uncoating and genome delivery, that are yet to be elucidated. At acidic pHs, these viruses appear to simply dissociate into subunits during uncoating, and the mechanisms by which they deliver their genomes across a cellular membrane into the cytoplasm are poorly understood. In contrast, the enterovirus capsid remains intact throughout the infection process, and models have been proposed for the mechanism by which these viruses interact with the membrane and deliver their genomes into the cytoplasm. However, it has yet to be established how broadly applicable these models are for all picornaviruses (30).We have recently shown that acid-induced capsid dissociation of ERAV proceeds via a transient intact empty particle, from which the RNA has been lost (31). This suggests a mechanism that coordinates genome release and delivery, as in the model for enteroviruses. To validate these studies in cell culture, we first wished to identify the endocytic route used by the virus and assess the role of acidification in the entry process. Picornaviruses utilize a variety of endocytic pathways. For example, FMDV and HRVs enter the cell via clathrin-mediated endocytosis and are subsequently delivered to the endosome. Here, they encounter an acidic pH, which is an indispensable step for a productive infection (2, 5, 12, 22). In contrast, echovirus 1 (enterovirus) uses the caveolin-dependent uptake of caveolae and delivery to caveosomes, where no change in pH is observed (16). PV entry is independent of clathrin- and caveolin-mediated endocytosis (6).To elucidate the entry route of ERAV, we used a combination of methods that include immunofluorescence (IF) microscopy, pharmacological inhibitors of specific endocytosis pathways, and dominant negative proteins.     

Characterization of a Putative Ancestor of Coxsackievirus B5

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.The group B coxsackieviruses (CVBs) (serotypes 1 to 6) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3D^pol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.     

Time-Dependent Transformation of the Herpesvirus Tegument

All herpesviruses have a layer of protein called the tegument that lies between the virion membrane and the capsid. The tegument consists of multiple, virus-encoded protein species that together can account for nearly half the total virus protein. To clarify the structure of the tegument and its attachment to the capsid, we used electron microscopy and protein analysis to examine the tegument of herpes simplex virus type 1 (HSV-1). Electron microscopic examination of intact virions revealed that whereas the tegument was asymmetrically distributed around the capsid in extracellular virions, it was symmetrically arranged in cell-associated virus. Examination of virions after treatment with nonionic detergent demonstrated that: (i) in extracellular virus the tegument was resistant to removal with Triton X-100 (TX-100), whereas it was lost nearly completely when cell-associated virus was treated in the same way; (ii) the tegument in TX-100-treated extracellular virions was asymmetrically distributed around the capsid as it is in unextracted virus; and (iii) in some images, tegument was seen to be linked to the capsid by short, regularly spaced connectors. Further analysis was carried out with extracellular virus harvested from cells at different times after infection. It was observed that while the amount of tegument present in virions was not affected by time of harvest, the amount remaining after TX-100 treatment increased markedly as the time of harvest was increased from 24 h to 64 h postinfection. The results support the view that HSV-1 virions undergo a time-dependent change in which the tegument is transformed from a state in which it is symmetrically organized around the capsid and extractable with TX-100 to a state where it is asymmetrically arranged and resistant to extraction.All herpesviruses have a tegument, a layer of protein located between the virus membrane and the capsid. Depending on the virus species, the tegument can be 20 to 40 nm in thickness, and it may be uniformly or asymmetrically distributed about the capsid (7, 17, 24, 33). The tegument is composed predominantly of virus-encoded proteins that together can account for up to half or more of the total virion protein mass. Tegument proteins are thought to be those involved in the early stages of infection before progeny virus proteins are synthesized.The tegument has been most thoroughly studied in herpes simplex virus type 1 (HSV-1). Examination of virions by electron microscopy has demonstrated that the tegument is not highly structured. Its morphology is described as predominantly granular with fibrous elements also present (7, 19). Analysis by cryo-electron microscopy, followed by icosahedral reconstruction has shown that the tegument is not icosahedrally ordered, although a small amount of tegument density is observed close to the capsid surface at the pentons (3, 47).The HSV-1 tegument is composed of approximately 20 distinct, virus-encoded protein species whose amounts vary considerably. The predominant components are UL47, UL48, and UL49, each of which occurs in more than 800 copies per virion (8, 46). In contrast, others, such as RL2 (ICP0), RS1 (ICP4), UL36, and UL37, occur in ∼100 copies or less. Trace amounts of host cell-encoded proteins are also present (15). Many of the tegument proteins are required for virus replication (34), and functions have been defined for most (9, 12, 31, 40).Biochemical studies have demonstrated that the tegument makes noncovalent contacts with both the virus capsid and the membrane. Studies of capsid-tegument contacts have emphasized binding of UL36, a tegument protein, to UL25, a capsid protein located near the vertices and involved in DNA encapsidation (5, 20, 29). Other tegument proteins such as UL48 (VP16), UL37, and UL49 (VP22) are found to associate with UL36 and may be bound to the capsid indirectly by way of UL36 (13, 44). UL16 binds reversibly to the capsid while UL46 (VP11/12) has been shown to bind to both the membrane and the capsid (21, 22, 26). Binding of tegument proteins to the membrane has been shown to occur by way of attachment to UL11 (45) and also to the internal domains of membrane glycoproteins, including glycoprotein D (gD), gH, and gE (4, 6, 11).We describe here the results of a study in which electron microscopy and protein analysis were used to clarify the structure of the HSV-1 tegument and its attachment to the capsid. The study was designed to extend the observation that most of the HSV-1 tegument remains attached to the capsid when the membrane is removed from the virus by treatment with nonionic detergent (19). Cell-associated and extracellular virions were compared after treatment with Triton X-100 (TX-100).     

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.     

Tryptophan Residues in the Portal Protein of Herpes Simplex Virus 1 Critical to the Interaction with Scaffold Proteins and Incorporation of the Portal into Capsids

Incorporation of the herpes simplex virus 1 (HSV-1) portal vertex into the capsid requires interaction with a 12-amino-acid hydrophobic domain within capsid scaffold proteins. The goal of this work was to identify domains and residues in the U_L6-encoded portal protein pU_L6 critical to the interaction with scaffold proteins. We show that whereas the wild-type portal and scaffold proteins readily coimmunoprecipitated with one another in the absence of other viral proteins, truncation beyond the first 18 or last 36 amino acids of the portal protein precluded this coimmunoprecipitation. The coimmunoprecipitation was also precluded by mutation of conserved tryptophan (W) residues to alanine (A) at positions 27, 90, 127, 163, 241, 262, 532, and 596 of U_L6. All of these W-to-A mutations precluded the rescue of a viral deletion mutant lacking U_L6, except W163A, which supported replication poorly, and W596A, which fully rescued replication. A recombinant virus bearing the W596A mutation replicated and packaged DNA normally, and scaffold proteins readily coimmunoprecipitated with portal protein from lysates of infected cells. Thus, viral functions compensated for the W596A mutation''s detrimental effects on the portal-scaffold interaction seen during transient expression of portal and scaffold proteins. In contrast, the W27A mutation precluded portal-scaffold interactions in infected cell lysates, reduced the solubility of pU_L6, decreased incorporation of the portal into capsids, and abrogated viral-DNA cleavage and packaging.Immature herpesvirus capsids or procapsids consist of two shells: an inner shell, or scaffold, and an outer shell that is roughly spherical and largely composed of the major capsid protein VP5 (24, 38).The capsid scaffold consists of a mixture of the U_L26.5 and U_L26 gene products, with the U_L26.5 gene product (pU_L26.5, ICP35, or VP22a) being the most abundant (1, 12, 20, 21, 32, 38). The U_L26.5 open reading frame shares its coding frame and C terminus with the U_L26 gene but initiates at codon 307 of U_L26 (17). The extreme C termini of both VP22a and the UL26-encoded protein (pU_L26) interact with the N terminus of VP5 (7, 14, 26, 40, 41). Capsid assembly likely initiates when the portal binds VP5/VP22a and/or VP5/pU_L26 complexes (22, 25). The addition of more of these complexes to growing capsid shells eventually produces a closed sphere bearing a single portal. pU_L26 within the scaffold contains a protease that cleaves itself between amino acids 247 and 248, separating pU_L26 into an N-terminal protease domain called VP24 and a C-terminal domain termed VP21 (4, 5, 8, 9, 28, 42). The protease also cleaves 25 amino acids from pU_L26 and VP22a to release VP5 (5, 8, 9). VP21 and VP22a are replaced with DNA when the DNA is packaged (12, 29).When capsids undergo maturation, the outer protein shell angularizes to become icosahedral (13). One fivefold-symmetrical vertex in the angularized outer capsid shell is biochemically distinct from the other 11 and is called the portal vertex because it serves as the channel through which DNA is inserted as it is packaged (23). In herpes simplex virus (HSV), the portal vertex is composed of 12 copies of the portal protein encoded by U_L6 (2, 23, 39). We and others have shown that interactions between scaffold and portal proteins are critical for incorporation of the portal into the capsid (15, 33, 44, 45). Twelve amino acids of scaffold proteins are sufficient to interact with the portal protein, and tyrosine and proline resides within this domain are critical for the interaction with scaffold proteins and incorporation of the portal into capsids (45).One goal of the current study was to map domains and residues within the U_L6-encoded portal protein that mediate interaction with scaffold proteins. We show that the portal-scaffold interaction requires all but the first 18 and last 36 amino acids of pU_L6, as well as several tryptophan residues positioned throughout the portal protein.     

Biological Effect of Muller's Ratchet: Distant Capsid Site Can Affect Picornavirus Protein Processing

Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21 cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical level the biological defects that arise when viruses are subjected to multiple bottleneck events.As a consequence of the quasispecies population structure, when a virus is subjected to an extreme bottleneck regime, such as successive plaque-to-plaque transfers, it accumulates deleterious mutations that result in fitness loss (reviewed in references 15, 21, and 33). These observations constitute experimental support for the Muller''s ratchet hypothesis, which states that asexual populations of organisms tend to acquire deleterious mutations unless compensatory mechanisms (such as sex or recombination) intervene (39, 41). Several lines of evidence indicate that population bottlenecks are abundant in the life cycle of viruses, both during host-to-host transmission and during intrahost replication (2, 8, 10, 22, 26, 32, 42, 45-48, 52, 53). Most studies have addressed the effects of bottlenecks on the reduction of intramutant spectrum diversity in relation to virus survival and persistence, effects on fitness, or as promoters of stochastic processes and drift in viral evolution. Yet the possible biological effects of specific mutations fixed as a result of bottleneck events remain largely unexplored.Experimental designs consisting of many successive plaque-to-plaque transfers, without intervening large-population passages, are ideal for obtaining viral clones that are debilitated by the occurrence of mutations because negative selection is highly attenuated (15, 21, 33). The deleterious nature of some mutations that become fixed in viral genomes subjected to repeated bottlenecks can be inferred from their position in the viral genome and then confirmed experimentally. For example, an internal tract of four oligoadenylate residues that precede the second functional AUG initiation codon of foot-and-mouth disease virus (FMDV) was invariant among natural isolates of the virus or among populations subjected to large-population passages. Yet this oligoadenylate tract was extended in several clones subjected to plaque-to-plaque transfers (17). This lesion, unique to clones that had undergone multiple bottleneck transfers, was associated with a decrease in replicative fitness (4, 17), and some of the clones displayed reduced levels of Lb, the form of the leader proteinase L synthesized from the second functional AUG initiation codon (17). However, the effect of other mutations that accumulate as a result of bottleneck transfers cannot be easily anticipated. Some mutations will likely be neutral while others are deleterious, and there is experimental and in silico evidence that a few mutations are advantageous or compensatory, thereby allowing the virus to survive despite continuous accumulation of mutations (21, 28).Nonsynonymous mutations in coding regions may perturb the structure and function of viral proteins. Despite evidence that such mutations can affect viral fitness, in very few cases the biochemical effect of a lesion associated with the operation of Muller''s ratchet has been identified. Here, we report that the accumulation of mutations in FMDV subjected to plaque-to-plaque passages results in a gradual increase in the thermosensitivity of infectious progeny production, with a several-logarithm decrease in progeny production at 42°C relative to 37°C at plaque transfer 230. Part of the thermosensitivity at early transfers could be traced to a single amino acid substitution, M54I, located at the B-C loop of capsid protein VP1. This loop corresponds to antigenic site 3 of FMDV (30, 36). We show that the M54I mutation decreases the proteolytic cleavage between capsid proteins VP3 and VP1 and that the impairment is manifested more severely at high temperatures. This cleavage is catalyzed by proteinase 3C^pro (31, 50, 51, 56), which does not show any substitution in the mutant FMDV clone that harbored the M54I mutation in VP1. Thus, a distant amino acid located at an antigenic site of a virus can affect a protein processing step catalyzed by wild-type 3C^pro. We discuss possible models to explain the link between two disparate phenotypic traits, antigenicity and protein processing, in the life cycle of a virus.     

Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3C^pro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10⁻³ nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5).Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53).Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3D^pol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events.To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.     

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.     

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.African swine fever virus (ASFV) (61, 72) is the only known DNA-containing arbovirus and the sole member of the Asfarviridae family (24). Infection by this virus of its natural hosts, the wild swine warthogs and bushpigs and the argasid ticks of the genus Ornithodoros, results in a mild disease, often asymptomatic, with low viremia titers, that in many cases develops into a persistent infection (3, 43, 71). In contrast, infection of domestic pigs leads to a lethal hemorrhagic fever for which the only available methods of disease control are the quarantine of the affected area and the elimination of the infected animals (51).The ASFV genome is a lineal molecule of double-stranded DNA of 170 to 190 kbp in length with convalently closed ends and terminal inverted repeats. The genome encodes more than 150 open reading frames, half of which lack any known or predictable function (16, 75).The virus particle, with an overall icosahedral shape and an average diameter of 200 nm (11), is organized in several concentric layers (6, 11, 15) containing more than 50 structural proteins (29). Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7, 34) on top of which the icosahedral capsid is assembled (26, 27, 31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11). Both forms of the virus, intracellular and extracellular, are infective (8).The assembly of ASFV particles occurs in the cytoplasm of the infected cell, in viral factories located close to the cell nucleus (6, 13, 49). ASFV factories possess several characteristics similar to those of the cellular aggresomes (35), which are accumulations of aggregates of cellular proteins that form perinuclear inclusions (44).Current models propose that ASFV assembly begins with the modification of endoplasmic reticulum (ER) membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. These ER-derived viral membranes represent the precursors of the inner viral envelope and are the first morphological evidence of viral assembly (7, 60). ASFV viral membrane precursors evolve into icosahedral intermediates and icosahedral particles by the progressive assembly of the outer capsid layer at the convex face of the precursor membranes (5, 26, 27, 31) through an ATP- and calcium-dependent process (19). At the same time, the core shell is formed underneath the concave face of the viral envelope, and the viral DNA and nucleoproteins are packaged and condensed to form the innermost electron-dense nucleoid (6, 9, 12, 69). However, the assembly of the capsid and the internal envelope appears to be largely independent of the components of the core of the particle, since the absence of the viral polyprotein pp220 during assembly produces empty virus-like particles that do not contain the core (9).Comparative genome analysis suggests that ASFV shares a common origin with the members of the proposed nucleocytoplasmic large DNA viruses (NCLDVs) (40, 41). The reconstructed phylogeny of NCLDVs as well as the similitude in the structures and organizations of the genomes indicates that ASFV is more closely related to poxviruses than to other members of the NCLDVs. A consensus about the origin and nature of the envelope of the immature form of vaccinia virus (VV), the prototypical poxvirus, seems to be emerging (10, 17, 20, 54). VV assembly starts with the appearance of crescent-shaped structures within specialized regions of the cytoplasm also known as viral factories (21, 23). The crescent membranes originate from preexisting membranes derived from some specialized compartment of the ER (32, 37, 52, 53, 67), and an operative pathway from the ER to the crescent membrane has recently been described (38, 39). VV crescents apparently grow in length while maintaining the same curvature until they become closed circles, spheres in three dimensions, called immature virions (IV) (22). The uniform curvature is produced by a honeycomb lattice of protein D13L (36, 70), which attaches rapidly to the membranes so that nascent viral membranes always appear to be coated over their entirety. The D13L protein is evolutionarily related to the capsid proteins of the other members of the NCLDV group, including ASFV, but lacks the C-terminal jelly roll motif (40). This structural difference is probably related to the fact that poxviruses are the only member of this group without an icosahedral capsid; instead, the spherical D13L coat acts as a scaffold during the IV stage but is discarded in subsequent steps of morphogenesis (10, 28, 46, 66). Thus, although crescents in VV and precursors of the inner envelope in ASFV are the first morphogenetic stages discernible in the viral factories of these viruses, they seem to be different in nature. Crescents are covered by the D13L protein and are more akin to the icosahedral intermediates of ASFV assembly, whereas ASFV viral membrane precursors are more similar to the naked membranes seen when VV morphogenesis is arrested by rifampin treatment (33, 47, 48, 50) or when the expression of the D13L and A17L proteins are repressed during infection with lethal conditional VV viruses (45, 55, 56, 68, 74, 76).Although available evidence strongly supports the reticular origin of the ASFV inner envelope (7, 60), the mechanism of acquisition remains unknown, and the number of membranes present in the inner envelope is controversial. The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7, 59, 60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope. In the case of VV, the coat of the D13L protein has been suggested to play a key role in the stabilization of the single membrane structure of the crescent (10, 17, 36), but the ASFV capsid protein p72 is not a component of the viral membrane precursors. The identification and functional characterization of the proteins involved in the generation of these structures are essential for the understanding of the mechanisms involved in these early stages of viral assembly. For this reason, we are focusing our interest on the study of abundant structural membrane proteins that reside at the inner envelope of the viral particle. We have shown previously that one of these proteins, p54, is essential for the recruitment of ER membranes to the viral factory (59). Repression of protein p54 expression has a profound impact on virus production and leads to an early arrest in virion morphogenesis, resulting in the virtual absence of membranes in the viral factory.Protein p17, encoded by the late gene D117L in the BA71V strain, is an abundant structural protein (60, 65). Its sequence, which is highly conserved among ASFV isolates (16), does not show any significant similarity with the sequences present in the databases. Protein p17 is an integral membrane protein (18) that is predicted to insert in membranes with a Singer type I topology and has been localized in the envelope precursors as well as in both intracellular and extracellular mature particles (60), suggesting that it resides at the internal envelope, the only membranous structure of the intracellular particles.In this work, we analyze the role of protein p17 in viral assembly by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-dependent lethal conditional virus. The data presented indicate that protein p17 is essential for viral morphogenesis. The repression of this protein appears to block assembly at the level of viral precursor membranes, resulting in their accumulation at the viral factory.From the electron microscopy analysis of serial sections of viral factories at very early times during morphogenesis, we present experimental evidence that suggests that, during assembly, viral precursor membranes and core material organize into large helicoidal intermediates from which icosahedral particles emerge. The possible role of these structures during ASFV morphogenesis is discussed.     

Effects of Major Capsid Proteins,Capsid Assembly,and DNA Cleavage/Packaging on the pUL17/pUL25 Complex of Herpes Simplex Virus 1

The U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pU_L17/pU_L25 interaction. We found that pU_L17 and pU_L25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the U_L19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pU_L17 (i) coimmunoprecipitated with pU_L25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pU_L25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pU_L25 in the absence of the triplex protein VP23 (encoded by the U_L18 gene), (iv) required pU_L25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pU_L25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pU_L25 in infected cell nuclei required pU_L17, pU_L32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pU_L15. The data suggest that VP23 or triplexes augment the pU_L17/pU_L25 interaction and that VP23 and VP5 induce conformational changes in pU_L17 and pU_L25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pU_L17/pU_L25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.Immature herpes simplex virus (HSV) capsids, like those of all herpesviruses, consist of two protein shells. The outer shell comprises 150 hexons, each composed of six copies of VP5, and 11 pentons, each containing five copies of VP5 (23, 29, 47). One vertex of fivefold symmetry is composed of 12 copies of the protein encoded by the U_L6 gene and serves as the portal through which DNA is inserted (22, 39). The pentons and hexons are linked together by 320 triplexes composed of two copies of the U_L18 gene product, VP23, and one copy of the U_L38 gene product, VP19C (23). Each triplex arrangement has two arms contacting neighboring VP5 subunits (47). The internal shell of the capsid consists primarily of more than 1,200 copies of the scaffold protein ICP35 (VP22a) and a smaller number of protease molecules encoded by the U_L26 open reading frame, which self-cleaves to form VP24 and VP21 derived from the amino and carboxyl termini, respectively (11, 12, 19, 25; reviewed in reference 31). The outer shell is virtually identical in the three capsid types found in HSV-infected cells, termed types A, B, and C (5, 6, 7, 29, 43, 48). It is believed that all three are derived from the immature procapsid (21, 38). Type C capsids contain DNA in place of the internal shell, type B capsids contain both shells, and type A capsids consist only of the outer shell (15, 16). Cleavage of viral DNA to produce type C capsids requires not only the portal protein, but all of the major capsid proteins and the products of the U_L15, U_L17, U_L28, U_L32, and U_L33 genes (2, 4, 10, 18, 26, 28, 35, 46). Only C capsids go on to become infectious virions (27).The outer capsid shell contains minor capsid proteins encoded by the U_L25 and U_L17 open reading frames (1, 17, 20). These proteins are located on the external surface of the viral capsid (24, 36, 44) and are believed to form a heterodimer arranged as a linear structure, termed the C capsid-specific complex (CCSC), located between pentons and hexons (41). This is consistent with the observation that levels of pU_L25 are increased in C capsids as opposed to in B capsids (30). On the other hand, other studies have indicated that at least some U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) associate with all capsid types, and pU_L17 can associate with enveloped light particles, which lack capsid and capsid proteins but contain a number of viral tegument proteins (28, 36, 37). How the U_L17 and U_L25 proteins attach to capsids is not currently known, although the structure of the CCSC suggests extensive contact with triplexes (41). It is also unclear when pU_L17 and pU_L25 become incorporated into the capsid during the assembly pathway. Less pU_L25 associates with pU_L17(−) capsids, suggesting that the two proteins bind capsids either cooperatively or sequentially, although this could also be consequential to the fact that less pU_L25 associates with capsids lacking DNA (30, 36).Both pU_L25 and pU_L17 are necessary for proper nucleocapsid assembly, but their respective deletion generates different phenotypes. Deletion of pU_L17 precludes DNA packaging and induces capsid aggregation in the nuclei of infected cells, suggesting a critical early function (28, 34), whereas deletion of pU_L25 precludes correct cleavage or retention of full-length cleaved DNA within the capsid (8, 20, 32), thus suggesting a critical function later in the assembly pathway.The current studies were undertaken to determine how pU_L17 and pU_L25 associate with capsids by studying their interaction and localization in the presence and absence of other capsid proteins.     

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.Human papillomaviruses (HPVs) exclusively infect cutaneous or mucosal epithelial tissues (14, 15, 30). HPV types that infect the mucosal epithelia can lead to the development of benign or malignant neoplasms, thus allowing for their categorization into low-risk or high-risk HPV types, respectively (14, 15, 30). A small subset of the more than 200 HPV types now identified are the causative agents of over 75% of all cervical cancers. HPV16 is the most prevalent type worldwide, found in ca. 50 to 62% of squamous cell carcinomas (14, 50).HPV16 virions contain a single, circular double-stranded DNA genome of ∼8 kb which associates with histones to form a chromatin-like structure. This minichromosome is packaged within a nonenveloped, icosahedral capsid composed of the major capsid protein L1 and the minor capsid protein L2. Similar to polyomaviruses, 72 capsomeres of L1 are geometrically arranged on a T=7 icosahedral lattice (2, 9, 17, 19, 36, 42). Recent cryoelectron microscopy images of HPV16 pseudovirions (PsV) suggest that L2 is arranged near the inner conical hollow of each L1 pentamer, although it is not known whether each L1 pentamer is occupied with a single L2 protein (5, 42).Due to technical constraints in the production of native HPV virions in organotypic culture, assembly studies of HPV particles have largely been restricted to the utilization of in vitro-derived particles such as virus-like particles (VLPs), PsV, and quasivirions (QV) (6, 12, 25, 40, 43). Recent research suggests that HPV and bovine papillomavirus PsV can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is characterized by resistance to proteolysis and chemical reduction and the appearance of a more orderly capsid structure via transmission electron microscopy (TEM) (7, 20).We present evidence that native virions, in the context of the complete papillomavirus life cycle, utilize a tissue-spanning redox gradient that facilitates multiple redox-dependent assembly and maturation events over the course of many days. We show that stability and specific infectivity of 20-day virions increases over 10-day virions, 20-day virions are more susceptible to neutralization than 10-day virions, and both viral DNA encapsidation and infectivity of HPV-infected tissues are redox dependent in that they can be manipulated via the treatment of organotypic tissues with oxidized glutathione (GSSG), which is concentration and temporally dependent.