Virology 254, 268–278 (1999) Article ID viro.1998.9548, available online at http://www.idealibrary.com on Hepatitis A Virus Translation Is Rate-Limiting for Virus Replication in MRC-5 Cells Ann W. Funkhouser,*,1 Derk E. Schultz,†,2 Stanley M. Lemon,† Robert H. Purcell,* and Suzanne U. Emerson* *Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892; and †Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019 Received July 13, 1998; returned to author for revision August 12, 1998; accepted November 30, 1998 Translation of hepatitis A virus (HAV) RNA is controlled by an internal ribosome entry site (IRES) located within the 59 untranslated region (UTR). In some cell types, the characteristically slow growth of HAV may be due to inefficient viral translation. We investigated whether this is true in MRC-5 cells, which are used for vaccine production. We measured the impact of two clusters of mutations in the 59 UTR on virus translation and replication: the AG group was selected during passage in African green monkey kidney cells, and the MR group was selected during subsequent passage in MRC-5 cells. The efficiency of cap-independent translation was assessed by inserting cDNA encoding an HAV IRES upstream of the chloramphenicol acetyl transferase gene and transcription was driven in vivo by a hybrid T7/vaccinia virus system. A luciferase gene was inserted upstream of the IRES to serve as an internal control. Each HAV UTR was also inserted into an infectious cDNA clone; the average rate of viral RNA accumulation was determined for each mutant virus. In MRC-5 cells, the rate of virus replication was highly correlated with the efficiency of cap-independent translation (P 5 0.006). The MR but not the AG mutations significantly increased both translation and viral RNA 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 correlation between the rate of virus replication and the efficiency of cap-independent translation (P 5 0.136): the AG and MR groups combined had a small impact on translation, but no detectable impact on virus replication. We conclude that in MRC-5 cells viral translation is rate-limiting for HAV replication. © 1999 Academic Press INTRODUCTION these mutations caused a maximum one-log increase in replication in BS-C-1 cells (Day et al., 1992). Schultz et al. Hepatitis A virus (HAV) is the only member of the (1996b) showed that these same mutations increase genus Hepatovirus in the family Picornaviridae. For rea- translation in BS-C-1 cells. Together, these published sons unknown, HAV replicates very inefficiently in cell data suggest a correlation between translation and HAV culture (Emerson et al., 1991). HAV also has an inefficient replication in BS-C-1 cells. In fetal rhesus kidney-4 internal ribosome entry site (IRES) compared with other (FRhK-4) cells, however, there are data suggesting that picornaviruses such as encephalomyocarditis virus (EMCV) (Whetter et al., 1994) and poliovirus (Schultz et HAV replication is not limited by the efficiency of virus al., 1996b). In vitro, replacement of the HAV 59 UTR with translation. Jia et al. (1996) found that insertion of an the more efficient EMCV IRES increases initiation at the efficient IRES (from EMCV) in front of the open reading correct polyprotein start site (Jia et al., 1991). However, it frame of HAV does not increase replication in FRhK-4 is unclear whether the extremely slow replication of HAV cells. In addition, Schultz et al. (1996b) found that muta- in cell culture is due to inefficient translation, to ineffi- tions that increase translation efficiency in BS-C-1 cells ciencies in other stages of the virus cycle, or to both. have no effect on translation efficiency in FRhK-4 cells. Viral translation limits the rate of viral replication in Slow growth and relatively low virus yields hamper many picornavirus systems (Simoes and Sarnow, 1991; production of HAV vaccine stocks in MRC-5 cells. Thus, Pilipenko et al., 1992; Todd et al., 1997). In the case of it is important to determine the restrictions to growth of hepatitis A virus, the importance of translation may re- HAV in this particular cell type. In one vaccine strain of flect the particular cells used. Day et al. (1992) showed HAV, we have discovered four naturally occurring muta- 9 that mutations at bases 152 and 203–4, and at base 687 tions in the 5 UTR clustered on a putative stem-loop in the 59 UTR, increase the radioimmunofocus size of before the translation start codon (A to G at position 591, HAV in BS-C-1 cells. Under one-step growth conditions, G to A at position 646, C to U at position 669, and U to G at position 687, Fig. 1). These mutations (the MR group) occurred when the HM175 strain of HAV was serially passaged in MRC-5 cells (human fetal lung fibroblasts) 1 To whom reprint requests should be addressed at present address: Section of Infectious Diseases, Department of Pediatrics, University of and are necessary for efficient replication of HM175 in Chicago, 5841 S. Maryland Avenue, Room C638B, Chicago, IL 60637. those cells (Funkhouser et al., 1994). The MR mutations 2 Present address: Qiagen Inc., Santa Clarita, CA 91355. have no discernible impact on virus replication in FRhK-4 0042-6822/99 $30.00 Copyright © 1999 by Academic Press 268 All rights of reproduction in any form reserved. HEPATITIS A VIRUS IN MRC-5 CELLS 269 FIG. 1. Diagram of putative secondary structure of the 59 untranslated region of hepatitis A virus (Brown et al., 1991). Mutations designated by black circles and nucleotide location; domains designated by Roman numerals; clusters of AG and MR mutations designated by boxes. cells (Funkhouser et al., 1996). Another group of muta- merase was provided by infection of cells with a recom- tions (U to C at position 124, deletion of bases 131 binant modified vaccinia virus Ankara (MVA/T7). The through 134 and 203, and A to G at position 152, Cohen translation efficiency of each mutant IRES was compared et al., 1987, Fig. 1) is also present in the 59 UTR of the to that of the wild-type in MRC-5 cells. BS-C-1 cells were MRC-5-adapted virus. These mutations (the AG group) also assayed because of the previous data showing that occurred during passage in African green monkey kid- HAV translation is correlated with radioimmunofocus ney (AGMK) cells and do not alter the rate of replication size in these cells (Day et al., 1992; Schultz et al., 1996b). in either MRC-5 or FRhK-4 cells (Funkhouser et al., 1994). Each IRES was incorporated into an infectious cDNA Since the MR mutations cause increased virus repli- clone derived from the MRC-5-adapted virus (pMR8, Fig. cation in MRC-5 cells, and all four of the MR mutations 2) and the effect of the mutated IRES on virus replication are included within the IRES, a likely hypothesis is that in either MRC-5 or BS-C-1 cells was determined. the MR mutations promote efficient replication of HAV in these cells, at least in part, by enhancing HAV transla- RESULTS tion. Because the MR mutations have such a marked Influence of MVA infection on CAT/LUC ratios cell-specific effect on replication of HAV in cultured cells (Funkhouser et al., 1996), study of these mutations pro- We confirmed that the differences noted in cap-inde- vides a unique opportunity to determine whether there is pendent translation between clones were not artifacts of a correlation between viral translation and replication. vaccinia infection. The impact of the MR mutations on We tested the hypothesis that the MR, but not the AG, cap-independent translation was tested in BT7-H cells in mutations increased viral translation in MRC-5 cells. the presence or absence of wild-type MVA. For each MRC-5 or BS-C-1 cells were transfected with bicistronic plasmid tested and in every experiment, the absolute plasmids to assay the efficiency of translation. The lucif- values of both CAT and LUC expression increased 500- erase (LUC) gene, driven by a T7 promoter, served as an to 1000-fold in the presence of vaccinia virus (data not internal control for translation of uncapped transcripts of shown). The cap-independent translations as measured the chloramphenicol acetyltransferase (CAT) gene. The by the CAT/LUC ratios of the plasmids with the most CAT gene was controlled by a HAV IRES containing the (pAGMR) and with the fewest (pWT) mutations were various naturally occurring mutations (Fig. 2). T7 poly- compared. Whereas the CAT/LUC ratio of the pAGMR 270 FUNKHOUSER ET AL. FIG. 2. Diagram of the bicistronic plasmids and HAV infectious cDNA clones. Designation of the bicistronic construct and infectious clone containing the same 59 UTR. IRES did not change significantly when vaccinia was RNA quantification added, that of pWT significantly increased in the pres- To be sure that any differences in CAT expression ence of vaccinia (Fig. 3). Therefore, vaccinia infection between different mutant RNAs reflected the level of decreased the range between mutant and wild-type cap-independent protein expression and not the level of clones, so that differences were underestimated. We transcription (Borman et al., 1994) directed by T7 ex- included MVA/T7 in our assay, nonetheless, since MRC-5 pressed from MVA, we assessed bicistronic RNA levels cells have a limited population doubling capacity and a by Northern analysis.