Molecular mechanisms of poliovirus persistence: key role of capsid determinants during the establishment phase

As viral persistence is of major medical importance, well-characterized, simple models are needed to improve our understanding of persistent infections. We have chosen to study the molecular mechanisms of viral persistence with the poliovirus (PV), because this picornavirus is one of the best characterized animal viruses, it infects the central nervous system which is a target organ for viral persistence, and it belongs to the Picornaviridae family of viruses, which includes several naturally persisting viruses. We have developed models of PV persistence in neuronal and epidermoid cells, and the present review will focus on the latter one because both lytic and persistent PV strains can be used to study the PV-HEp-2 cell interactions. The viral determinants of persistence have been investigated with this model, and PV determinants have proven to be of crucial importance for the establishment of persistence in HEp-2 cells. Precise determinants of PV persistence have been identified for PV serotypes 1 and 3, in capsid proteins VP1 and VP2. These determinants modify the early steps of the PV cycle, and in particular, the conformational modifications of the capsid following virus adsorption onto its receptor. These results permit us to propose several hypotheses concerning PV persistence and the early steps of the PV cycle.     

Randomized comparison of early and late vaccination with inactivated poliovirus vaccine after allogeneic BMT

Forty-five adult HLA-matched sibling BMT recipients were randomized to receive inactivated poliovirus vaccine (IPV) at 6, 8 and 14 months (early group, n = 23) or at 18, 20 and 26 months after BMT (late group, n = 22). Ninety-five percent of the early group patients had protective antibody titres of > or = 4 to poliovirus type 1 (PV1), poliovirus type 2 (PV2) and poliovirus type 3 (PV3) by a microneutralization assay prior to the first vaccination, at 6 months after BMT. The corresponding proportion for the late group patients was only 67% at 18 months. The antibody responses 1 month after each of the three IPV doses were similar in the two vaccination groups, except that four-fold responses occurred more frequently after the first dose to PV2 and PV3 in the late group. All patients had a protective antibody titre to all poliovirus serotypes 1 and 22 months after the third vaccine dose, except one patient in the early group who lacked antibodies to PV3 at 22 months. Acute GVHD accelerated the decrease of poliovirus antibody titres prior to vaccination but had no influence on vaccination response. Chronic GVHD neither influenced the patient's ability to retain poliovirus antibodies prior to vaccination nor impaired responses to vaccinations. A vaccination schedule consisting of three IPV doses was equally immunogenic when started at 6 or 18 months after allogeneic BMT.     

The poliovirus empty capsid specifically recognizes the poliovirus receptor and undergoes some, but not all, of the transitions associated with cell entry

Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled.     

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution

BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins

A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 x 10(6) infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.     

Potential use of poliovirus as a vector

The live attenuated Sabin strains of poliovirus have proven their efficacy at inducing a good humoral and secretory antibody response in humans. The extensive characterization of poliovirus neutralization antigenic sites and the atomic resolution of the three-dimensional structure of the viral capsid have enabled the use of the most stably attenuated poliovirus strain (the Sabin type 1 strain) as a vector for the presentation of short foreign antigenic domains in place of one of its own neutralization antigenic sites. The creation of such chimeras has been achieved by manipulating poliovirus infectious cDNA and transfecting the resulting chimeric cDNAs onto susceptible cell cultures. However, this epitope-presentation system has a limitation in terms of the sequence and size of the foreign domain that can be incorporated into the poliovirus capsid without disrupting virus viability. This has led to the construction of poliovirus hybrid genomes bearing insertions of longer heterologous sequences in place of part of the poliovirus structural genes. Upon transfection onto susceptible cells providing the poliovirus structural proteins in trans (e.g. cells previously infected with the Sabin 1 strain), stocks of encapsidated RNA replicons which expressed the foreign protein could be obtained. In addition, viable recombinant viruses bearing insertions of heterologous sequences at various places into the poliovirus genome without deleting poliovirus sequences have been reported. Potential applications of these chimeric and recombinant polioviruses in the engineering of new recombinant vaccines are discussed.     

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.     

Profiles of subjective quality of life in schizophrenic in- and out-patient samples

The genus Enterovirus is a large group of viruses belonging to the family Picornaviridae. They have a worldwide distribution, cause a wide spectrum of disease, and are a common cause of central nervous system disease. Included among the sixty-six enterovirus serotypes known to infect humans are the three poliovirus (PV) serotypes, the cause of paralytic poliomyelitis (PPM). PPM has been controlled in many parts of the world by vaccination. Molecular and functional comparison of PV vaccine strain and wild neurovirulent PV strains has provided an insight into mechanisms of neurovirulence. Enteroviruses are also the most commonly implicated viral agents of aseptic meningitis. Less commonly they cause a more serious encephalitis. Specific antiviral treatment for enterovirus infections is not currently available. Virological diagnosis is nonetheless important to distinguish between enterovirus-induced meningitis or encephalitis and other treatable causes of disease with a similar clinical picture.     

Identification of regions of poliovirus 2BC protein that are involved in cytotoxicity

The expression of poliovirus 2BC protein in yeast and mammalian cells leads to a number of metabolic and morphological alterations, such as growth inhibition, intracellular membrane proliferation, blockade of the exocytic pathway, and enhanced membrane permeability. Yeast cells that express poliovirus 2BC in an inducible manner were used to identify the regions of 2BC implicated in the modifications of these cellular functions. Several 2BC deletion mutants were generated to define the minimal portion of 2BC required to alter these activities. Additional deletion mutants that were obtained by random mutagenesis followed by selection in yeast cells provided new insights into the structure and mechanism of action of 2BC. The activity responsible for membrane proliferation is located in 2C, while the activities responsible for membrane permeabilization and inhibition of the exocytic pathway are located in 2B. Several regions of 2B and 2C required for the different functions of 2BC were identified. Thus, the integrity of the N termini of both 2B and 2C is necessary for 2BC-induced cytotoxicity. It is also possible to separate the different cellular alterations provoked by 2BC by the use of several 2BC variants. Deletion of amino acids 52 to 65 in 2B generates a 2BC deletion variant, 2bC deltaAvrII, that still blocks yeast growth but is unable to enhance membrane permeability or to inhibit the exocytic pathway. On the other hand, 2Bcl28*.32b and 2Bcl28*.3c, which contain only 73 and 77 amino acids of 2B, interfere with yeast division and enhance membrane permeability but affect the exocytic pathway only weakly and do not induce membrane proliferation. Our findings indicate that Saccharomyces cerevisiae represents a useful model system to analyze the functions of poliovirus 2BC and show the feasibility of separating the activities assigned to this protein.     

Placental transfer of maternal poliovirus antibodies in full-term and pre-term infants

This study was designed to investigate the placental transfer of maternal poliovirus antibodies in full-term and pre-term infants. Two hundred healthy, Israeli born mothers and their infants, were enrolled immediately after birth. The study population comprised two groups: a full-term group of 150 mothers and their infants, and a pre-term group of 50 mothers and their infants (gestational age < 35 weeks). Maternal and umbilical cord blood samples were taken in all cases. Antibody titers against the three poliovirus serotypes and a polio virus type 1 strain that caused an outbreak in 1988 (epidemic strain 1) were measured by a microneutralization system. The proportion of individuals with protective titers against each of the poliovirus types tested was slightly lower in the infants compared with their mothers. When protection to all strains combined was tested, the difference between mothers and infants was significant (P < 0.05). Transplacental transfer to epidemic strain 1 was less effective--12% of the premature infants were not protected against it at birth. The geometric mean titers against poliovirus types 1, 3 and epidemic type 1 strain were significantly lower in the pre-term group than in the full-term group. In both the full-term and pre-term groups there were significant linear correlations between the maternal and neonatal antibody titers for each of the polio viruses tested. For all poliovirus types, the transfer of maternal antibodies to the full-term infant was significantly higher than the transfer of maternal antibodies to the pre-term infant (P < 0.001). Owing to diminished transfer of maternal antibodies, pre-term infants are at greater risk of poliovirus infection.     

A protein linkage map of the P2 nonstructural proteins of poliovirus

The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.     

The crystal structure of bacteriophage Q beta at 3.5 A resolution

BACKGROUND: The capsid protein subunits of small RNA bacteriophages form a T = 3 particle upon assembly and RNA encapsidation. Dimers of the capsid protein repress translation of the replicase gene product by binding to the ribosome binding site and this interaction is believed to initiate RNA encapsidation. We have determined the crystal structure of phage Q beta with the aim of clarifying which factors are the most important for particle assembly and RNA interaction in the small phages. RESULTS: The crystal structure of bacteriophage Q beta determined at 3.5 A resolution shows that the capsid is stabilized by disulfide bonds on each side of the flexible loops that are situated around the fivefold and quasi-sixfold axes. As in other small RNA phages, the protein capsid is constructed from subunits which associate into dimers. A contiguous ten-stranded antiparallel beta sheet facing the RNA is formed in the dimer. The disulfide bonds lock the constituent dimers of the capsid covalently in the T = 3 lattice. CONCLUSIONS: The unusual stability of the Q beta particle is due to the tight dimer interactions and the disulfide bonds linking each dimer covalently to the rest of the capsid. A comparison with the structure of the related phage MS2 shows that although the fold of the Q beta coat protein is very similar, the details of the protein-protein interactions are completely different. The most conserved region of the protein is at the surface, which, in MS2, is involved in RNA binding.     

Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscopy and X-ray crystallography of virus-Fab complexes

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the beta-B-beta-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid

We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range.     

The crystal structure of bluetongue virus VP7

Bluetongue virus (BTV), a representative of the orbivirus genus of the Reoviridae, is considerably larger (at 80 nm across), and structurally more complex, than any virus for which we have comprehensive structural information. Orbiviruses infect mammalian hosts through insect vectors and cause economically important diseases of domesticated animals. They possess a segmented double-stranded RNA genome within a capsid composed of four major types of polypeptide chains. An outer layer of VP2 and VP5 is removed as the virus enters the target cell, to leave an intact core within the cell. This core is 70 nm across and composed of 780 copies of VP7 (M(r) 38K) that, as trimers, form 260 'bristly' capsomeres clothing an inner scaffold constructed from VP3 (M(r) 103K). We report here the crystal structure of VP7 from BTV serotype 10, which reveals a molecular architecture not seen previously in viral structural proteins. Each subunit consists of two domains, one a beta-sandwich, the other a bundle of alpha-helices, and a short carboxy-terminal arm which might tie trimers together during capsid formation. A concentration of methionine residues at the core of the molecule could provide plasticity, relieving structural mismatches during assembly.