RESUMO
Poliovirus mutants were selected during the persistent infection of human neuroblastoma cells. These viruses could establish secondary persistent infections in HEp-2 nonneural cells. We report the identification of a region of the genome of a persistent virus (S11) that was sufficient to confer to a recombinant virus the phenotype that causes persistent infection in HEp-2 cells. This region, between nucleotides 1148 and 3481, contained 11 missense mutations mapping exclusively in the genes of capsid proteins VP1 and VP2. Because recombinant viruses carrying only one of these two mutated genes were not able to cause persistent infection, it seems very probable that two or more mutations in these genes are required for expression of the phenotype that causes persistent infection.
Assuntos
Genes Virais , Poliovirus/crescimento & desenvolvimento , Proteínas Estruturais Virais/genética , Capsídeo/genética , Proteínas do Capsídeo , Células Cultivadas , DNA Recombinante , Humanos , Técnicas In Vitro , Poliovirus/genética , Replicação ViralRESUMO
Poliovirus mutants selected in persistently infected human neuroblastoma cells have a modified cell tropism and can establish a secondary persistent infection in nonneural cells, such as HEp-2c cells. Nucleotide sequence analysis revealed that the genome of a persistent mutant, S11, differed from that of the parental lytic Sabin 1 poliovirus strain by 31 point mutations. Three mutations occurred in the noncoding regions. The other mutations resulted in 12 amino acid substitutions; 1 substitution occurred in a nonstructural protein (3A), while the other 11 substitutions were clustered in the capsid proteins VP2 and VP1. The same missense mutations, as well as many of the silent mutations that we observed in mutant S11, also accumulated in the genome of two other persistent viruses isolated from independent infections. This finding indicates that both missense and silent mutations are selected during the persistent infection of neuroblastoma cells and suggests that the secondary structure of RNA in the coding region may play a role in viral infection.
Assuntos
Mutação , Poliovirus/genética , Capsídeo/genética , Proteínas do Capsídeo , Genoma Viral , Humanos , Especificidade de Órgãos , Mutação Puntual , Poliovirus/patogenicidade , Sequências Reguladoras de Ácido Nucleico/genética , Seleção Genética , Análise de Sequência , Células Tumorais CultivadasRESUMO
Mutants of poliovirus (PV) with highly modified biological properties can be selected in vitro in cells of neural origin. Mutations accumulate in the genome of type 1 PV strains selected in human neuroblastoma cells, modifying cell specificity and conferring to the virus the ability to persist in such nonneural cells as HEp-2c (Pelletier et al., Virology 180, 729 1991). With this cell system, we have both parent lytic strains and persistent PV mutants; these were used to study the mechanisms of the establishment and maintenance of the persistent infection. We found that a persistent infection was established when the lytic potential of the virus was reduced; this involved both an early and a late event of the virus cycle for the type 1 mutants. In contrast, maintenance of the infection did not correlate with the reduced lytic potential of the viruses, but rather with the selection of mutant cell populations of various phenotypes. Two cell lines, representative of two phenotypes, were studied in greater detail. In the first one, HEp-S32 (cl7), the PV receptor was not detected by cytofluorometry and viral genomes were detected by in situ hybridization in 2% of the cells. In the second cell line, HEp-S31 (cl18), 97% of the cells expressed the PV receptor, viral genomes were detected in 9-10% of the cells, and viral antigens in 5-10% of the cells. With this cell line, the cure of the culture or, alternatively, the lysis of the majority of cells, could be induced under specific culture conditions. We propose a model involving an equilibrium between an abortive and a lytic infection to explain the properties of cells persistently infected with PV.
Assuntos
Poliovirus/fisiologia , Animais , Northern Blotting , Linhagem Celular , Imunofluorescência , Fluorometria , Genoma Viral , Humanos , Cinética , Camundongos , Neuroblastoma , Hibridização de Ácido Nucleico , Fenótipo , Poliovirus/genética , Poliovirus/crescimento & desenvolvimento , Temperatura , Fatores de Tempo , Células Tumorais Cultivadas , Replicação ViralRESUMO
Six Sabin-derived persistent poliovirus mutants were selected in human neuroblastoma IMR-32 cells. The mutants had a titer 30 to 10(5) times lower in nonneural HEp-2c cells than in IMR-32 cells. When the growth cycles of persistent viruses in the two cell lines were compared, the most striking feature was a delay of 2 to 4 hr in virus release from HEp-2c cells. In Hep-2c cells, type 1 mutants could spontaneously establish a persistent infection in the absence of any exogenous viral inhibitor. Mutations at a rate of 1 every 210 nucleotides had accumulated in the genome of the type 1 mutants selected in neuroblastoma cells, modifying cell specificity and conferring the ability to persist in some nonneural cells. These results indicate that mutants of poliovirus with highly modified biological properties can be selected in vitro in cells of neural origin.
