Hepatitis A virus translation is rate-limiting for virus replication in MRC-5 cells

Translation of hepatitis A virus (HAV) RNA is controlled by an internal rib osome entry site (IRES) located within the 5' untranslated region (UTR). In some cell types, the characteristically slow growth of HAV may be due to i nefficient viral translation. We investigated whether this is true in MRC-5 cells, which are used for vaccine production. We measured the impact of tw o clusters of mutations in the 5' UTR on virus translation and replication: the AG group was selected during passage in African green monkey kidney ce lls, and the MR group was selected during subsequent passage in MRC-5 cells . The efficiency of cap-independent translation was assessed by inserting c DNA encoding an HAV IRES upstream of the chloramphenicol acetyl transferase gene and transcription was driven in vivo by a hybrid T7/vaccinia virus sy stem. A luciferase gene was inserted upstream of the IRES to serve as an in ternal control. Each HAV UTR was also inserted into an infectious cDNA clon e; the average rate of viral RNA accumulation was determined for each mutan t virus. In MRC-5 cells, the rate of virus replication was highly correlate d with the efficiency of cap-independent translation (P = 0.006). The MR bu t not the AG mutations significantly increased both translation and viral R NA accumulation. Reversion of just one MR mutation (687 G to A) eliminated all of the replication-stimulating and translation-enhancing effects of the MR mutations. In the control BS-C-1 cells, there was no discernible correl ation between the rate of virus replication and the efficiency of cap-indep endent translation (P = 0.136): the AG and MR groups combined had a small i mpact on translation, but no detectable impact on virus replication. We con clude that in MRC-5 cells viral translation is rate-limiting for HAV replic ation, (C) 1999 Academic Press.