![]() Here, using a phage display selection, we obtained two antibody fragments (Fabs), HCV2 and HCV3, against HCV IRES that bind the RNA with dissociation constants of 32 ± 7 nM and 37 ± 8 nM respectively, specifically recognizing the so-called junction IIIabc (JIIIabc). Structured RNA elements within the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome hijack host cell machinery for translation initiation through a cap-independent mechanism. Dots under a nucleotide within boxes A and B indicate identical nucleotide matches between HCV-1 and BVDV. The two domains of HCV-1 with significant similarity to bovine viral diarrhea virus (BVDV) are boxed (spaces between adjacent nucleotides required for optimal alignment of HCV-1 and BVDV are omitted in this figure). The closed triangle indicates the position of an insertion of two nucleotides seen in isolate HK2, whereas the open triangle indicates the position of insertions of a single nucleotide seen in isolates HK2, Z5, and Z8. Uppercase and lowercase letters indicate nucleotide positions that are invariant and variable, respectively. Open bars represent contribution of published sequences closed bars represent data from this study. The consensus sequence and the percent of sequences divergent from it are given at each nucleotide position for all sequences available at that site. The sequence of all 282 nucleotide positions was available in 53 isolates, of which 39 were from this study the remaining sequences were partial sequences. Histogram of the percent of sequences different from the consensus sequence at each nucleotide position in 282 nucleotides of the 5' noncoding region among 81 HCV isolates (37 published sequences and 44 sequences from this study) from around the world. These data also have implications for selection of optimal primer sequences for the detection of HCV RNA by the PCR assay. Our sequence analysis of the 5' NC region of the HCV genome provides additional information about conserved elements within this region and suggests a possible functional role for the region in viral replication or gene expression. Additional analysis revealed the presence of short open reading frames in all HCV isolates. Two nucleotide domains within the 5' NC region, conserved among all HCV isolates studied to date, shared statistically significant similarity with pestivirus 5' NC sequences, providing further evidence for a close evolutionary relationship between these two groups of viruses. Conversely, there were three highly conserved domains consisting of 18, 22, and 63 completely invariant nucleotides (positions -263 to -246, -199 to -178, and -65 to -3, respectively). Nucleotide variations were found in 45 (16%) of the 282 nucleotide positions analyzed and were primarily located in three domains of significant heterogeneity (positions -239 to -222, -167 to -118, and -100 to -72). The consensus sequence was identical to the prototype HCV sequence. Analysis of the nucleotide sequence from 44 HCV isolates in this study combined with that of 37 isolates reported in the literature reveals that the 5' NC region of HCV consists of highly conserved domains interspersed with variable domains. Nucleotide insertions were seen in three isolates. The most distantly related isolates were only 90.1% identical. We have identified several HCV isolates with significantly greater sequence heterogeneity than reported previously within the 5' NC region. We have determined the nucleotide sequence of the 5' noncoding (NC) region of the hepatitis C virus (HCV) genome in 44 isolates from around the world. ![]()
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