Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome

Abstract

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5°C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.

Emergency Services of Viral RNAs: Repair and Remodeling

“…5). Interestingly, engineered mutant polioviruses with similar extended deletions proved to be markedly attenuated in the monkey neurovirulence assay (328).…”

The 5′-Untranslated Region of Picornaviral Genomes

“…This stretch may perform an essential function, as first suggested by Kuge and Nomoto (1987) on the basis of the assessment of the viability of poliovirus deletion mutants. This theory was further supported by experiments with modified poliovirus (Iizuka et al, 1989) and coxsackievirus B1 (Iizuka et al, 1990;A. Nomoto and N. Iizuka, personal communication) 5-UTRs.…”

“…Specifically, the removal of nucleotides 630-726, 622-726, or 600-726 from the POliovirus type 1 (Sabin) genome does not alter such in uitro phenotypic properties of the virus as the plaque size or the time course of virus growth and development of cytopathic changes (see also Trono et al, 1988a). More extended deletions, 570-726 or 564-726, also produce viable progeny, but the mutant viruses exhibit a small-plaque (BPI phenotype due to their retarded growth rate (see also Iizuka et al, 1989). Assuming that phenotypic changes in this series of mutants are largely due to the altered function of the cis-acting translation control element (and we believe that this is a likely assumption), it could be concluded that the downstream border of this element should map between positions 564 and 600.…”

“…Moreover, these experiments showed directly that mutations within this stretch did affect the translation efficiency. Curiously, the sequence with coordinates 539-563 in the poliovirus type 1 RNA exhibits a striking complementarity to sequence 1301-1320 in human 28 S rRNA (Iizuka et al, 1989); the significance of this observation remains entirely obscure.…”

“…Moreover, Bienkowska-Szewczyk and inferred from their in uitro experiments with truncated templates that the corresponding sequence would be essential for the internal initiation translation. Nevertheless, the fact that the removal of nucleotides 564-726 (i.e., deletion of the entire domain E) from poliovirus RNA or the corresponding segment from coxsackievirus B 1 RNA did not abrogate the virus viability (but only retarded its growth) Iizuka et al, 1989Iizuka et al, ,1990 indicates, perhaps, unambiguously that this domain is not essential for the virus-specific translation. Some additional information about the significance of its structural details is presented in Section V,C, and speculations about its specific role are considered in Section X.…”