Structural Requirements for Initiation of Translation by Internal Ribosome Entry within Genome-Length Hepatitis C Virus RNA

Cap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5' nontranslated RNA (5'NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5'NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5'NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5'NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translation in vitro in rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28-69 of the 5'NTR (stem-loop IIa) sharply reduced capsid translation both in vitro and in vivo. A small deletion mutation involving nt 328-334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation both in vitro and in vivo, while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5'NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation

Natural Variation in Translational Activities of the 5′ Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site

The 5′ nontranslated RNA (5′NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5′NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31–42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5′NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5′NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K(+) concentrations, with a sevenfold difference evident at 130 to 150 mM K(+). These results suggest that there is an RNA-RNA interaction involving 5′NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N

Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule

Sequence Requirements for Viral RNA Replication and VPg Uridylylation Directed by the Internal cis-Acting Replication Element (cre) of Human Rhinovirus Type 14

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5′ and 3′ noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5′ half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation

Size Heterogeneity in the 3′ Noncoding Region of South American Isolates of Yellow Fever Virus

The 3′ noncoding region (3′ NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3′ NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3′ NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-ΔRYF). To investigate the biological significance of these 3′ NCR rearrangements, YF-XL-ΔRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-ΔRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3′ NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti