Two determinants in the capsid of a persistent type 3 poliovirus exert different effects on mutant virus uncoating.

Mutant polioviruses (PV) have been previously found to be capable of establishing persistent infections in HEp-2c cells. Together, two amino acid substitutions in the viral capsid of a type 3 poliovirus (PV-3), at positions VP213 and VP1(290), are sufficient to confer the persistent phenotype to a normally lytic virus. When susceptible cells are infected, the double mutant T7L+2L(13)1N(290) undergoes unique conformational changes in the capsid, modifying its sedimentation coefficient from 160S to 147S. In the present study, we have further investigated mutant PV decapsidation and, in particular, the effect of each determinant independently. Our results indicate that the novel 147S form was also generated by a mutant carrying only the determinant 1N290. This form was not produced as a result of inherent capsid instability and it was generated only upon specific PV-host cell interactions. The second viral determinant, 2L13, also modified receptor-induced conformational changes, although differently from 1N290

Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions.

After 2.5 months of persistent infection in human neuroblastoma cells by the type 3 poliovirus (PV3) wild-type Leon strain, a mutant (PVpi), L2-2, capable of establishing a persistent infection in nonneural HEp-2c cells was isolated. Sequence analysis of the viral capsid protein genes revealed the presence of seven missense mutations, three of which were also present in a second PVpi, suggesting that they could be important determinants of the persistent phenotype. When the three mutations were introduced into the lytic Leon strain separately, in pairs or all together, all but one of the viruses was capable of establishing a persistent infection. However, aside from the triple mutant, only one mutant virus, bearing a Leu at position VP213 in the capsid interior and an Asn at position VP1290 on the capsid surface, was capable of establishing persistent infections in more than 30% of the cultures. When present together, these two determinants affect the early steps of the virus cycle including cell binding and the receptor-mediated conformational changes believed to be necessary for viral penetration and uncoating. In fact, this persistent double mutant appears to undergo a novel capsid transition when in contact with the human PV receptor, altering from the native virion which sediments at 160S to a form which sediments at about 147S. We propose that this modification could be the mechanism by which PV3 is able to establish persistent infections in HEp-2c cell cultures

Genetic stability of poliovirus insertion mutants with a foreign oligopeptide on the capsid surface.

The genetic stability of poliovirus mutants which carry a foreign oligopeptide on the surface of their capsid was studied (1) upon mutant isolation, (2) after serially diluted passages in cell cultures, and (3) in persistently infected cultures which have been recently developed. Viruses having a 3-codon insertion within the VP1 capsid protein-encoding region appeared to be extremely stable, except in the specific case of persistent infection. Viruses having a 6-codon insertion were slightly less stable. Point mutations and one recombination event were observed as soon as viruses were recovered and studied following plasmid transfection. Additional point mutations appeared within the insertion after 12 serially diluted passages in monkey kidney cells. Under all test conditions, the foreign insertion was never deleted from the virus genome

One amino acid change on the capsid surface of poliovirus Sabin 1 allows the establishment of persistent infections in HEp-2c cell cultures.

Poliovirus mutants (PVpi) selected during the persistent infection of human neuroblastoma cells can establish secondary persistent infections in nonneural HEp-2c cells (I. Pelletier, T. Couderc, S. Borzakian, E. Wyckoff, R. Crainic, E. Ehrenfeld, and F. Colb?-Garapin, 1991, Virology, 180, 729-737). Previous results from our laboratory have also shown that, in the genome of PVpi S11 derived from the Sabin 1 strain, the genomic region involved in this phenotype contains 11 missense mutations which map exclusively to the genes encoding the capsid proteins VP1 and VP2. We report here the identification of precise viral determinants able to confer the capacity to establish persistent infections in HEp-2c cell cultures to the lytic Sabin 1 strain. We used a strategy based on the observation that PVpi, after a few months of persistent infection in HEp-2c cells, tend to regain a more lytic phenotype in uninfected HEp-2c cell cultures. We constructed mutant viruses carrying only a few mutations potentially involved in the phenotype of persistence. Two mutations were identified, one corresponding to the substitution His>Tyr of amino acid 142 of VP2 and another corresponding to the substitution Val>Ile of amino acid 160 of VP1. Mutants carrying one or the other of the two determinants established persistent infections in HEp-2c cell cultures in about 20% of the infections. Higher frequencies were obtained with the mutant carrying both determinants (30%), and with PVpi S11 (63%), indicating that the effects of several determinants can be cumulative. The two determinants are localized on the capsid surface in a region known to be involved in the interactions between poliovirus and its cell receptor and in fact, we demonstrate here that in the case of the two persistent mutants, these interactions are modified

Persistent poliovirus infection of human fetal brain cells.

It has been suggested that poliovirus (PV), the causative agent of poliomyelitis, could persist in surviving patients. We have previously shown that PV can persistently infect some human cell lines in vitro, particularly neuroblastoma cell lines. We report here an ex vivo model in which PV can persistently infect primary cultures of human fetal brain cells. Two mutations involving capsid residues 142 of VP2 and 95 of VP1 were repeatedly selected during the persistent infections. These residues are located in capsid regions known to be involved in interactions between PV and its receptor. During the first week after infection, viral antigens were found in cells of both the neuronal and glial lineages. In contrast, 2 weeks after infection, viral antigens were detected almost exclusively in cells of the neuronal lineage. They were detected predominantly in cells expressing a marker of early commitment to the neuronal lineage, MAP-5, particularly in neuroblasts. Viral antigens were also found in immature progenitors expressing a neuroepithelium marker, nestin, and in cells expressing a marker of postmitotic neurons, MAP-2. The presence of viral antigens in postmitotic neurons suggests that PV can persist in neurons of patients who have survived poliomyelitis

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

Persistent poliovirus infection of human neuroblastoma cells.

Two human neuroblastoma cell lines were persistently infected with poliovirus strains of all three serotypes. In persistently infected IMR-32 cells, which were studied in greatest detail, viral antigens were present in most cells, and over a 9-month period virions were found in the medium at high titers. Persistently infected cells were resistant to superinfection by Sabin 1, 2, and 3 poliovirus but sensitive to coxsackievirus B3. The viruses recovered from persistently infected cells were studied for conservation of epitopes, host cell specificity, and temperature resistance phenotype. The antigenic site 1 carried by the major capsid protein VP1 was modified on the persistent viruses of all three serotypes. This was confirmed for one virus by sequencing the corresponding genomic region in which two mutations were detected. The titers of persistent viruses were 1-3 log10 units higher on IMR-32 cells than on nonneuronal HEp-2 cells, while parental viruses had similar titers on both lines. When thermosensitive viruses were used to initiate the infection, the persistent viruses were found to be thermoresistant at 39 degrees C. Together the results indicate that the persistent infection correlated with the selection of highly mutated viral strains. Poliovirus-infected neuroblastoma cell lines thus constitute an in vitro model of chronic viral infections, which are increasingly implicated in human neural diseases