The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.

Although the lengthy 5' nontranslated regions (5'NTRs) of other picornaviral RNAs form highly ordered structures with important functions in viral translation, little is known about the 5'NTR of hepatitis A virus (HAV). We determined the nearly complete 5'NTR nucleotide sequences of two genetically divergent HAV strains (PA21 and CF53) and included these data in a comparative phylogenetic analysis of the HAV 5'NTR. We identified covariant nucleotide substitutions predictive of conserved secondary structures and used this information to develop a model of the 5'NTR secondary structure, which was further refined by thermodynamic predictions and nuclease digestion experiments. According to this model, the 5'NTR comprises six major structural domains. Domains I and II (bases 1 to 95) contain a 5'-terminal hairpin and two stem-loops followed by a single-stranded and highly variable pyrimidine-rich tract (bases 96 to 154). The remainder of the 5'NTR (domains III to VI, bases 155 to 734) contains several complex stem-loops, one of which may form a pseudoknot, and terminates in a highly conserved region containing an oligopyrimidine tract preceding the putative start codon by 13 bases. To determine which structural elements might function as an internal ribosome entry site, RNA transcripts representing the HAV 5'NTR with progressive 5' deletions were translated in rabbit reticulocyte lysates. The translation product was truncated, unprocessed P1 polyprotein. Removal of the 5'-terminal 354 bases of the 5'NTR had little effect on translation. However, deletion to base 447 slightly decreased translation, while deletion to base 533 almost completely abolished it. These data indicate that sequences 3' of base 355 play an important role in the translation mechanism utilized by genomic-length HAV RNA. Significantly, this region shares several conserved structural features with the internal ribosome entry site element of murine encephalomyocarditis virus.

To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)

The lengthy 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) forms a highly ordered secondary structure, which has been suggested to play an important role in controlling viral translation by allowing for translation initiation by internal ribosome entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the HAV 5'NTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron designed to block ribosomal scanning, the HAV 5'NTR was capable of directing the internal initiation of translation, confirming the presence of an internal ribosome entry site (IRES). Analysis of various deletion mutants demonstrated that the 5' border of the IRES is located between nucleotides 151 and 257, while the 3' border extends to the 3' end of the 5'NTR, between nucleotide 695 and the first initiation codon at 735. Except for a segment between bases 638 and 694, deletion of stem-loop structures between bases 151 and the 3' end of the 5'NTR inhibited or abolished translation. The addition of a 5' cap structure (m7GpppN) to monocistronic or bicistronic transcripts decreased the translation of a reporter gene downstream of the HAV 5'NTR but enhanced translation of the upstream cistron in bicistronic transcripts. This finding indicates that a 5' cap structure is inhibitory to HAV IRES-directed translation initiation and that the cap structure and the HAV IRES probably compete for the same limiting translation factors. The efficiency with which monocistronic constructs containing the HAV 5'NTR directed translation in reticulocyte lysates was compared with results for monocistronic constructs containing the IRES of the more rapidly growing encephalomyocarditis virus (EMCV). These results indicated that the HAV 5'NTR was more than 25-fold less active than the EMCV IRES in producing translation product. HAV 5'NTR-directed translation was inhibited by the presence of a one-fifth molar quantity of RNA containing the EMCV IRES, while a fivefold molar excess of the HAV 5'NTR did not inhibit EMCV IRES-directed translation. The relatively weak activity of the HAV IRES may thus be due to a reduced affinity for cellular translation factors which are present in limiting quantities in rabbit reticulocyte lysate.

The 5' nontranslated RNA (5'NTR) of the HM175 strain of human hepatitis A virus contains several pyrimidine-rich regions, the largest and most 5' of which (pY1) is an almost pure polypyrimidine tract located between nucleotides (nt) 99 and 138, which includes five tandem repeats of the sequence motif (U)UUCC(C). Previous modeling of the RNA secondary structure suggested that this region was likely to be single-stranded, but repetitive RNase V1 cleavage sites within these (U)UUCC(C) motifs indicated that pY1 possesses an ordered structure. To assess the role of this domain in replication of the virus, a series of large deletion mutations were created which involved the pY1 domain of an infectious cDNA clone. Deletion of 44 nt between nt 96 and 139, including the entire pY1 domain, did not reduce the capacity of the virus to replicate in BS-C-1 or FRhK-4 cells, as assessed by the size of replication foci in radioimmunofocus assays or by virus yields under one-step growth conditions. In contrast, viable virus could not be recovered from transfected RNAs in which the deletion was extended in a 5' direction by an additional 3 nt (delta 93-134), most likely because of the destabilization of a predicted stem-loop structure upstream of pY1. Deletion mutations extending in a 3' fashion to nt 140, 141, or 144 resulted in moderately (delta 96-140 and delta 96-141) or strongly (delta 99-144, delta 116-144, and delta 131-144) temperature-sensitive replication phenotypes. Although deletion of the pY1 domain did not by itself affect the replication phenotype of virus, the additional deletion of sequence elements within the pY1 domain (nt 99 to 130) substantially enhanced the temperature-sensitive phenotype of delta 131-144 virus. These data suggest that the (U)UUCC(C) motifs within the pY1 domain are conserved among wild-type viruses in order to serve a function required during infection in vivo but not in cell culture. In contrast, the single-stranded region located immediately downstream of pY1 (nt 140 to 144) is essential for efficient replication in cultured cells at physiological temperature. Viruses with deletion mutations involving nt 140 to 144 and viruses with large pY1 deletions but normal replication phenotypes in cell culture may have attenuation properties which could be exploited for vaccine development.