Large deletion mutations involving the first pyrimidine-rich tract of the 5' nontranslated RNA of human hepatitis A virus define two adjacent domains associated with distinct replication phenotypes

Abstract

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.

Replication of hepatitis A viruses with chimeric 5' nontranslated regions

“…Similar pseudoknot structures have been predicted for members of the cardiovirus and aphthovirus groups (24). Immediately downstream, there is a pyrimidine-rich sequence, called pY1, which extends from nt 99 to 138 and contains a series of tandem repeats of a U 3 C 3 sequence motif that are stacked into an ordered structure that is resistant to single-strand-specific RNases (33). Surprisingly, this region could be deleted from the viral RNA with no apparent effect on virus replication in BS-C-1 or FRhK-4 cells (33).…”

“…The 5Ј NTR of picornaviral RNA contains sequences and structural motifs required for RNA replication and ribosome binding and possibly for other functions, such as RNA packaging and assembly of virus particles. Mutational mapping studies and structural analyses conducted predominantly by the Lemon laboratory (7,8,33) have generated a model which is schematically depicted in Fig. 7.…”

“…Immediately downstream, there is a pyrimidine-rich sequence, called pY1, which extends from nt 99 to 138 and contains a series of tandem repeats of a U 3 C 3 sequence motif that are stacked into an ordered structure that is resistant to single-strand-specific RNases (33). Surprisingly, this region could be deleted from the viral RNA with no apparent effect on virus replication in BS-C-1 or FRhK-4 cells (33). Extension of the deletion downstream, past nt 139 into an apparently single-stranded region (nt 140 to 153), resulted in virus replication defects.…”

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine

“…It is not clear how the 5Ј NC mutations affect either virulence or growth in cell culture. The AGMK-specific mutations are located between bases 124 and 207 near a poly(C ⅐ U) tract (35) and may overlap the internal ribosome entry site (20,23), the 5Ј end of which may start somewhere between bases 151 and 251 (3,19). Interestingly, deletion of the poly(C ⅐ U) tract from base 96 to base 137 had no apparent effect on viral replication in cell culture or in tamarins (35), suggesting that only very particular changes in the RNA secondary structure in this region of the genome have a significant effect on the phenotype.…”

“…The AGMK-specific mutations are located between bases 124 and 207 near a poly(C ⅐ U) tract (35) and may overlap the internal ribosome entry site (20,23), the 5Ј end of which may start somewhere between bases 151 and 251 (3,19). Interestingly, deletion of the poly(C ⅐ U) tract from base 96 to base 137 had no apparent effect on viral replication in cell culture or in tamarins (35), suggesting that only very particular changes in the RNA secondary structure in this region of the genome have a significant effect on the phenotype. One or more of the AGMK-specific mutations have a cell-specific effect on growth efficiency; they are required for efficient growth in CV-1 cell culture but not in FRhK-4 cell culture (16), and the mutation at base 152 in this group also increases growth in BS-C-1 cells (11).…”