Identification of a novel sequence at the 3' end of the GB virus B genome

Abstract

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.

FIG. 1

Oligonucleotides used in this study. (Top) Schematic representation of the GBV-B genome showing the orientations and approximate positions of the primers. (Bottom) Locations and sequences of the primers. Numbering refers to the GBV-B genomic sequence, including the C at position 9138, the 3′Y (259 nt), and a poly(U) tract of 27 nt, as in the U22304 sequence.

FIG. 2

Sequence of the 3′ UTR of the GBV-B genome following the poly(U) tract. nt 9138 and 9145 to 9403 (3′Y), not present in the published sequence of the GBV-B genome (GenBank accession no. U22304), are underlined. For numbering, see the legend to Fig. 1.

FIG. 3

Detection of the 3′Y sequence in GBV-B-infected tamarin liver RNA and lack of detection of the 3′Y sequence in noninfected tamarin genomic DNA. (A) Northern blot of tamarin liver RNA. RNA samples: lanes 1 and 4, uninfected tamarin RNA; lanes 2 and 5, GBV-B-infected S. fuscicollis tamarin RNA; lanes 3 and 6, GBV-B-infected S. oedipus tamarin RNA. DNA probes: lanes 1 to 3, 3′Y probe; lanes 4 to 6, control probe. (B) Northern blot of GBV-B-infected S. oedipus tamarin RNA. RNA probes: lane 1, 3′Y-antisense; lane 2, 3′Y-sense; lane 3, control-antisense; lane 4, control-sense. The solid arrows indicate the viral genomic RNA band; the open arrows indicate the low-molecular-weight RNA species. (C) Southern blot of noninfected S. oedipus tamarin genomic DNA. Lane 1, GBV-B 3′Y probe; lane 2, GBV-B control probe; lane 3, tamarin gene probe.

FIG. 4

Comparison of the putative secondary structures of the 3′-end regions of GBV-A, GBV-B, and HCV encompassing the 3′ stem-loop structures (3′-SL I, 3′-SL II, and 3′-SL III). In 3′-SL II of GBV-A and GBV-B, identical nucleotides are indicated in white on circular black backgrounds. GBV-B and HCV structures start at the nucleotide following poly(U); GBV-A, lacking a poly(U) tract, starts at a position chosen to highlight the similarity with the GBV-B structure. RNA secondary structure prediction was performed by running the program Mfold of the Wisconsin Package, version 9.1 (Genetics Computer Group [GCG], Madison, Wis.). The output was produced by the program Plotfold of the GCG package.