Temperature-sensitive hepatitis A virus mutants with deletions downstream of the first pyrimidine-rich tract of the 5' nontranslated RNA are impaired in RNA synthesis.

Hepatitis A virus (HAV) mutants containing large deletions within the first pyrimidine-rich tract (pY1; nucleotides [nt] 99 to 138) of the 5' nontranslated RNA (5'NTR) replicate well in cultured cells, while those with pY1 deletions which extend in a 3' direction to include nt 140 to 144 (CUUGU) have a temperature-sensitive (ts) replication phenotype (D.R. Shaffer, E.A. Brown, and S.M. Lemon, J. Virol. 68:5568-5578, 1994). To characterize this replication defect, the ts mutant delta 131-144 was grown under one-step conditions at the nonpermissive temperature (37 degrees C). A shift to the permissive temperature (31 degrees C) for the first 18 h of the viral replication cycle did not enhance virus yields, indicating that temperature sensitivity is not due to a defect in viral entry or uncoating. Similarly, absence of increased yield with a late shift to 31 degrees C between 54 and 72 h suggested that the ts defect does not involve viral assembly. Although monocistronic RNA transcripts containing the delta 99-144 deletion directed translation 22 to 58% less efficiently than the standard 5'NTR in transfected BS-C-1 cells, this difference was present at both 31 and 37 degrees C. In addition, there were no temperature-dependent differences in the abilities of bicistronic transcripts containing either ts or non-ts 5'NTR sequences within the intercistronic space to direct translation of a downstream reporter gene. Thus, ts mutations do not confer a demonstrable temperature-related defect in cap-independent translation. In contrast, an RNase protection assay showed that synthesis of viral plus-strand RNA was markedly delayed in BS-C-1 cells infected with ts virus at 37 degrees C. Analysis of the nucleotide sequence surrounding the deletion in a non-ts revertant derived from delta 116-144 virus revealed that a single U-to-G transversion at nt 114 (CUUUU-->CUUGU) had restored the sequence normally present between nt 140 and 144. These results indicate that ts mutants of HAV with deletions extending downstream from the pY1 domain to nt 140 to 144 are defective in RNA synthesis and that the single-stranded RNA segment containing nt 140 to 144 plays a critical role in replication of HAV RNA.