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.     

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.     

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.     

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.     

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.     

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.     

Systematic Study of the Genetic Response of a Variable Virus to the Introduction of Deleterious Mutations in a Functional Capsid Region

We have targeted the intersubunit interfaces in the capsid of foot-and-mouth disease virus to investigate the genetic response of a variable virus when individual deleterious mutations are systematically introduced along a functionally defined region of its genome. We had previously found that the individual truncation (by mutation to alanine) of 28 of the 42 amino acid side chains per protomer involved in interactions between capsid pentameric subunits severely impaired infectivity. We have now used viral RNAs individually containing each of those 28 deleterious mutations (or a few others) to carry out a total of 96 transfections of susceptible cells, generally followed by passage(s) of the viral progeny in cell culture. The results revealed a very high frequency of fixation in the capsid of second-site, stereochemically diverse substitutions that compensated for the detrimental effect of primary substitutions at many different positions. Most second-site substitutions occurred at or near the capsid interpentamer interfaces and involved residues that are spatially very close to the originally substituted residue. However, others occurred far from the primary substitution, and even from the interpentamer interfaces. Remarkably, most second-site substitutions involved only a few capsid residues, which acted as “second-site hot spots.” Substitutions at these hot spots compensated for the deleterious effects of many different replacements at diverse positions. The remarkable capacity of the virus to respond to the introduction of deleterious mutations in the capsid with the frequent fixation of diverse second-site mutations, and the existence of second-site hot spots, may have important implications for virus evolution.The rapid replication, high mutation rates, and large population sizes of RNA viruses give rise to genetically highly heterogeneous virus populations, termed viral quasispecies (15-18). Viral quasispecies may quickly adapt to different environments through the fixation of some of the multiple mutations present in individual genomes in the population. Accordingly, fixation of second-site mutations able to compensate for the effect of randomly occurring deleterious mutations could be particularly frequent in RNA virus populations. Compensatory mutations have indeed been detected, many times in a fortuitous way, during genetic analyses of different viruses (4, 6, 7, 9, 12, 13, 22, 29, 33, 36-42, 48, 49, 51-54). However, to our knowledge no study has systematically analyzed the frequency, types, and distribution of second-site mutations acquired by a virus in response to many different deleterious mutations introduced along a extended, but functionally defined, region of its genome.The viral capsid provides a particularly interesting target to analyze the fixation of second-site, compensatory mutations in a functional region of a virus. Nonenveloped virus capsids may be subjected to multiple, severe, and even contradictory selective pressures imposed on them to preserve their adaptive physicochemical properties and multiple biological functions (8, 31). Accordingly, accumulating evidence indicates that the capsids of small, nonenveloped viruses have been exquisitely fine-tuned through evolution, probably to a much larger extent than most cellular proteins or protein complexes. Structure-function analyses almost invariably show that the vast majority of amino acid replacements in nonenveloped viral capsids, even in exposed loops with no known function, significantly decreases viral yields, or at least unfavorably affects viral fitness (see, for example, references 28, 31, 32, and 43 and references therein). Thus, despite the dramatic potential for variation in viral quasispecies, strong negative selection could be expected to lead to very high amino acid sequence conservation in the capsid proteins. In fact, such a high sequence conservation is not observed for many nonenveloped RNA virus capsids. This paradox could be resolved by invoking a frequent fixation in the capsid region of compensatory second-site mutations, which could help the virus to evade the immune response and to adapt to new environments and hosts.Foot-and-mouth disease virus (FMDV) is a small, icosahedral, nonenveloped RNA virus whose structure, function, and evolution have been widely studied (27, 46). FMDV populations are quasispecies (19), and the FMDV capsid may provide a good model for the study of compensatory mutations and their role in virus variation and evolution. In addition, this virus is the causative agent of foot-and-mouth disease (FMD), one of the economically most important animal diseases worldwide. Frequent genetic and antigenic variation in the capsid proteins impose severe difficulties for FMD control (27, 31, 46); thus, a profound knowledge of the dynamics of compensatory mutations in the FMDV capsid may also contribute to improve current strategies to fight FMD and other diseases caused by highly variable viruses.The FMDV capsid is formed by 60 copies of each of three proteins (VP1, VP2, and VP3) and a small, internal polypeptide (VP4). During assembly of FMDV and other picornaviruses, one copy of the precursor polyprotein P1 folds and is proteolytically processed to yield a protomeric subunit composed of one copy of each capsid protein. Five protomers oligomerize to yield a pentameric subunit, and twelve pentamers assemble to form an icosahedral capsid (44) (Fig. ​(Fig.1A).1A). The three-dimensional structure of several FMDV isolates (1, 11, 21, 26), including variant C-S8c1, a biological clone from vaccine strain C₁Santa Pau-Spain/70, is known (26). Based on this structural information, we carried out previously a systematic structure-function study of the capsid interpentamer interfaces in FMDV C-S8c1. The analysis revealed that most interfacial side chains were critically required for viral function and infectivity (28). However, partial sequencing fortuitously revealed the occasional fixation in the capsid of a few pseudoreversions and second-site amino acid substitutions that, in some cases, were responsible for restoring the infectivity, thus acting as compensatory mutations (28). These and other observations suggested the possibility that a complex dynamics of compensatory mutations could be operating at the functionally critical intersubunit interfaces during the evolution of FMDV.Open in a separate windowFIG. 1.Primary and second-site substitutions in the FMDV capsid. (A) Schematic quaternary structure of the capsid. Each protein subunit is represented by a trapezoid. The numbers 1, 2, and 3, respectively, denote VP1, VP2, and VP3. The black pentagons, triangles, and ellipses indicate the positions of capsid fivefold, threefold, and twofold symmetry axes, respectively. Three protomeric subunits around a threefold axis, each of them belonging to a different pentameric subunit, are colored deep red, yellow, or blue. Three adjacent pentamers to which those protomers belong are colored in shades of red, yellow, or blue, respectively. (B and C) Primary and second-site substitutions, mapped on a partial model of the FMDV C-S8c1 capsid structure (26). The image represents a wireframe atomic model of the three protomers in the scheme shown in panel A that belong to adjacent pentamers and are deep-colored and labeled, plus one additional VP2 subunit (also labeled in panel A) from each of the three pentamers. A threefold axis is located at the center of the image, and the color code is as in panel A. The interfaces between three pentamers are thus defined by the limits between different colors (compare also panel A). In panel B, the interfacial residues subjected to deleterious substitutions are shown as white space-filling models. In panel C, the capsid residues where second-site substitutions were partially or totally fixed in the viral progeny populations are shown as space-filling models; they are colored either green (residues involved in second-site substitutions that were dominant in the population) or orange (residues involved in nondominant, “third-site” substitutions).In the present study we have systematically analyzed the frequency, types, and patterns of second-site amino acid substitutions fixed in the FMDV capsid in response to the introduction of intrinsically deleterious substitutions of the functionally critical residues at the interpentamer interfaces. The results have revealed a complex dynamics of very frequent and diverse second-site mutations, many of them concentrated in a few capsid hot spots. A working model to integrate the large amount of data obtained on the frequency, type, location and other characteristics of the second-site mutations detected is discussed.     

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.     

Early Steps in Cell Infection by Parvoviruses: Host-Specific Differences in Cell Receptor Binding but Similar Endosomal Trafficking

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are closely related parvoviruses that differ in their host ranges for cats and dogs. Both viruses bind their host transferrin receptor (TfR), enter cells by clathrin-mediated endocytosis, and traffic with that receptor through endosomal pathways. Infection by these viruses appears to be inefficient and slow, with low numbers of virions infecting the cell after a number of hours. Species-specific binding to TfR controls viral host range, and in this study FPV and strains of CPV differed in the levels of cell attachment, uptake, and infection in canine and feline cells. During infection, CPV particles initially bound and trafficked passively on the filopodia of canine cells while they bound to the cell body of feline cells. That binding was associated with the TfR as it was disrupted by anti-TfR antibodies. Capsids were taken up from the cell surface with different kinetics in canine and feline cells but, unlike transferrin, most did not recycle. Capsids labeled with fluorescent markers were seen in Rab5-, Rab7-, or Rab11-positive endosomal compartments within minutes of uptake, but reached the nucleus. Constitutively active or dominant negative Rab mutants changed the intracellular distribution of capsids and affected the infectivity of virus in cells.Cell infection by animal viruses involves a specific sequence of steps that deliver the virus and its genome from the cell surface to the compartment where replication can occur. For nonenveloped viruses, infection initiates with binding to a specific cell receptor and uptake into the cell by receptor-mediated endocytosis. Various factors can control the process of viral uptake, including the characteristics of the receptor(s) bound by the virus and its signaling and endocytic properties, the affinity of the virus for the receptor, and the structural features of the interaction in different environments (36, 61). Receptors may be located on the cell body or may also be displayed on the extended lamellipodia or filopodia with greater surface areas. Viruses binding to filopodia can be either passively delivered to the cell body for endocytosis by dynamic movement of the entire structure or actively trafficked by retrograde actin transport as well as the action of myosin-2 motors on the actin (32, 57). Cross-linking and clustering of receptors by viral particles can influence the rate and pathways of uptake from the cell surface (23), and many viral receptors activate signaling pathways that alter the structure of the underlying cytoskeleton to enhance uptake (see, e.g., references 12, 30, and 51). Receptor-bound viruses then enter one or more endosomal pathways that results in the capsid being enclosed in vesicles and trafficked within the endosomal pathways of the cell, where clustered virus and receptors (23) may undergo structural alterations upon exposure to conditions such as low pH or proteases (36, 61). The specific receptor-mediated binding and entry pathways often provide signals for viruses that allow endosomal escape and establish infection. A variety of markers of the endosomal compartments have been used in studies of viral entry. Rab proteins are monomeric small GTPases which regulate endosomal membrane trafficking, and specific Rab proteins are associated with different endosomal compartments. Among the many Rab proteins in the cells, Rab5 is primarily associated with the early endosome and regulates trafficking through that compartment, Rab7 is associated with the late endosome, and Rab11 is associated with the recycling endosome (14, 58). Tracking viral particles within the endosomal pathways during cell entry has been used to define the steps in the entry and infection processes of a variety of different viruses and has revealed many of the common features and variant processes that are used (7-9, 33, 71).Here, we examine the uptake and infection of cells by parvovirus capsids and compare some of the steps followed by capsids that differ in their receptor binding properties and host ranges. Feline panleukopenia virus (FPV) infects cats (50, 66), binds the transferrin receptor-1 (TfR) on feline cells, and uses that receptor for uptake and infection (27, 44). FPV does not bind the canine TfR or infect dogs or cultured canine cells. Canine parvovirus (CPV) is a natural variant of FPV which emerged in 1978 after acquiring a small number of mutations that allow its capsid to bind the canine TfR (27). The original strain of CPV (designated CPV type 2 [CPV-2]) spread worldwide in dogs during 1978, but some of the same mutations that gave it the canine host range rendered it unable to infect cats (66, 67). CPV-2 was replaced worldwide during 1979 and 1980 by a natural variant, CPV type 2a (CPV-2a), which contained an additional four to five changes in its coat protein gene (48, 49). Subsequently, the canine viruses have continued to evolve, and additional single mutations have been selected that alter antigenic epitopes. Strains altered at VP2 residue 426 are designated CPV-2b (Asn426Asp) and CPV-2c (Asp426Glu) (13, 48). CPV-2a and its variants are able to infect both dogs and cats but show reduced binding to the feline TfR on cells and in vitro (27, 42). In addition, the affinity of binding to the canine TfR is much lower than that seen for the feline TfR (42).The TfR is a type II membrane protein expressed in nonlipid raft regions of the plasma membrane, and it binds iron-loaded (holo) transferrin (Tf) at neutral pH (2). TfR expression is tightly regulated, and it is more highly expressed on dividing cells with high iron needs, which would favor binding of these viruses. The TfRs of mice and humans are used as receptors for cell infection by the mouse mammary tumor virus and the New World hemorrhagic fever arenaviruses (52, 56).The TfR is assembled as a homodimer, and each monomer of the ectodomain is composed of protease-like, apical and helical domains, as well as a 30-Å membrane-proximal stalk (5, 20, 31). The transmembrane domain mediates membrane insertion and influences some aspects of trafficking within the cell, while the cytoplasmic domain contains a tyrosine-threonine-arginine-phenylalanine (YTRF) sequence that engages the clathrin-mediated endocytic machinery through AP-2 (adaptor protein-2) (53, 55). The TfR sequence also includes one or two cysteines adjacent to the inner leaflet of the membrane that may be palmitoylated to influence the rate of receptor recycling, and it also contains sequences that control basolateral localization in polarized cells (41). In the normal pathway of TfR-mediated entry, the TfR-holo-Tf complex is transported into the endosomal system, where low pH results in conformational changes and iron release. The TfR-Tf complex enters the early endosome, from which some of the complex is rapidly recycled to the cell surface while most passes to the perinuclear recycling endosome. From there it recycles to the cell surface where the iron-free apo-Tf is released at neutral pH (21, 22, 24, 37, 38, 69, 70). The rate of uptake and the efficiency of TfR recycling depend on the form of the ligand, and more than 97% of monomeric Tf recycles to the cell surface within 10 to 30 min. However, cross-linking TfRs with oligomeric Tf or antibodies causes the complexes to be retained within endosomes for longer times, and a higher proportion is trafficked to late endosomes and lysosomes for degradation (35).Holo-Tf binds the membrane-proximal side of the feline and canine TfR ectodomain (11), while virus binding involves the apical domain of the receptor as mutations in that structure affect the ability to bind FPV and CPV capsids (43) The feline and canine TfRs differ in ∼10% of their sequences, but a major difference controlling the CPV-specific binding is a unique glycosylation site in the apical domain of the canine TfR (43). Alteration of the glycosylated Asn to Lys (the feline TfR residue) allowed the canine TfR to bind FPV and also greatly increased the affinity of binding to CPV-2 and CPV-2a-related capsids (42).CPV and FPV have small (25 nm) nonenveloped capsids that package a single-stranded DNA genome of ∼5,120 bases (68). The particles are made up of two overlapping proteins, VP1 and VP2, with 90% of the capsid protein being VP2. VP1 contains a 143-residue amino (N)-terminal sequence that encodes a phospholipase A₂ enzymatic activity, as well as basic amino acid motifs that play a role in nuclear localization (72). The VP1 unique region becomes exposed during cell entry without capsid disintegration, and the phospholipase A₂ modifies the endosomal membrane to enhance endosomal escape (19, 75).Previous studies of cell entry by CPV, minute virus of mice, and various adeno-associated viruses (AAVs) show that viral uptake primarily occurs through clathrin-mediated endocytosis. However, when the AP-2-interacting sequences in the cytoplasmic tail of the feline TfR were mutated or deleted, the altered receptor still allowed CPV infection at a similar efficiency to that of wild-type TfR (26). The intracellular pathways of viral entry and trafficking have been examined by using cells fixed at various times after uptake and then antibody stained for virus and cellular markers or by expressing green fluorescent protein (GFP)-labeled markers. Time courses examined were between 1 and 6 h, and sequential steps of trafficking were suggested, with the virus passed from the early endosomes to the recycling endosome, followed by localization in late endosomes and lysosomes after uptake (65). By fluorescent antibody staining, VP1 release occurred only hours after uptake, possibly in a low-pH degradative compartment (64, 72). In addition, CPV capsids appear to remain associated with the receptor for 1 to 2 h after virus uptake as antibodies against the TfR cytoplasmic tail microinjected into feline CRFK cells block infection in this time period (44). Infection is also blocked by neutralizing the low pH of the endosomal system with ammonium chloride or bafilomycin A1, although it is not clear whether this is due to direct effects on the capsid or to indirect alterations in endosomal trafficking. When the X-ray crystal structures of capsids of CPV and FPV were determined at low pH or in the presence of EDTA or when capsids were examined for changes in protease susceptibility, only small changes in surface loops of the viral structure were present (40, 60).Here, we used microscopy to examine dynamic steps in the binding, uptake, and early trafficking of parvovirus capsids in live canine and feline cells. Labeled capsids were seen to undergo rapid movement into multiple endosomal compartments shortly after entry. Initial binding of CPV to canine cells involved filopodia while in feline cells the virus bound primarily to receptors on the cell body. In cells expressing GFP-conjugated Rab proteins, particles rapidly localized to multiple endosomal compartments in the cytoplasm after uptake, which gradually accumulated near the microtubule-organizing center. The distribution of intracellular viruses and the viral infectivity in feline cells were altered by expression of either constitutively active (CA) or dominant negative (DN) mutants of the Rab proteins.     

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).     

A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.Virus infections depend on complex interactions between viral and cellular proteins. Consequently, the nature of these interactions has important implications for viral cell type specificity, tissue tropism, and pathogenesis. Group B coxsackieviruses (CVB1 to CVB6), members of the genus Enterovirus within the family of Picornaviridae, are human pathogens that cause a broad spectrum of diseases, ranging from mild upper respiratory illnesses to more severe infections of the central nervous system, heart, and pancreas (61). These viruses have also been associated with certain chronic muscle diseases and myocardial infarction (2, 3, 12, 13, 22).The positive single-stranded RNA genome (approximately 7,500 nucleotides in length) of CVBs is encapsidated within a small T=1, icosahedral shell (30 nm in diameter) comprised of repeating identical subunits made up of four structural proteins (VP1 to VP4). Parts of VP1, VP2, and VP3 are exposed on the outer surface of the capsid, whereas VP4 is positioned on the interior. The virion morphology is characterized by a star-shaped mesa at each 5-fold icosahedral symmetry axis, surrounded by a narrow depression referred to as the “canyon” (69). All six serotypes of CVB can use the coxsackie and adenovirus receptor (CAR) for cell attachment and entry (9, 55, 82). Some strains of CVB1, -3, and -5 also use decay accelerating factor ([DAF] CD55) for initial attachment to the host cell; however, binding to DAF alone is insufficient to permit entry into the cell (10, 54, 76).Picornaviruses are generally characterized by their cytolytic nature in cell culture. However, several in vivo and in vitro studies have shown that some picornaviruses, e.g., poliovirus, Theiler''s murine encephalomyelitis virus, foot-and-mouth disease virus, CVB3, CVB4, and CVB5, may also establish persistent, noncytolytic infections (4, 29, 35, 39, 62, 74). Recently, it has been shown that the diverse outcomes of picornaviral infections may depend on interactions between the virus and the apoptotic machinery of the infected cell (14, 30, 71). Several picornaviral proteins have been identified as inducers of an apoptotic response, including viral capsid proteins VP1, VP2, and VP3, as well as nonstructural proteins 2A and 3C (7, 20, 32, 33, 42, 50, 63). In addition, antiapoptotic activity has been assigned to the nonstructural proteins 2B and 3A (16, 59).Picornaviruses have the potential to adapt rapidly to new host environments. Virus features affecting adaptability include high mutation rates, short replication times, large populations, and frequent incidences of recombination (25-27, 53). Consequently, picornaviruses exist as genetically heterogenous populations, referred to as viral quasispecies (25, 26).Previously, the CVB2 prototype strain Ohio-1 (CVB2O) was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells (66). Two amino acid changes were identified in the capsid-coding region, and one was identified in the 2C-coding region of the adapted virus. Further characterization of the virus-host interaction showed that the infection was not affected by anti-DAF antibodies, indicating the use of an alternative receptor.In this study, the amino acid substitutions associated with the adaptation of CVB2O to cytolytic infection of RD cells were evaluated. Site-directed mutagenesis studies showed that a single amino acid change in the VP1 capsid protein was responsible for the cytolytic RD phenotype. In addition, as indicated by caspase activation and DNA degradation, the apoptotic pathway was activated in RD cells infected by the cytolytic virus.     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

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.