J. T. Stapleton and others Fig. 1. Genome organization of the GB viruses and HCV. All four viruses contain a positive-polarity ssRNA genome with a 59 NTR and 39 NTR. The genome encodes a polyprotein that is co- and post-translationally cleaved into individual viral proteins. The structural proteins include core (C) and envelope glycoproteins (E1 and E2), and the NS proteins include NS2–NS5B. The presence of a genomic coding region for a C protein has not been identified for GBV-A or GBV-C. Structural proteins are cleaved by cellular signal peptidases (open arrows), and the NS2–NS3 cleavage is accomplished by the NS2–NS3 autoprotease (shaded arrows). The remaining NS proteins are cleaved by the NS3–NS4A protease complex (solid block arrows). The predicted genome organization of GBV-D was based on a polyprotein starting nt 18 of GU566735. *, The predicted sizes of the proteins analogous to the HCV p7 are 21 kDa for GBV-A and 6 kDa for GBV-C. The existence of a GBV- D p7-like protein is not clear from sequence analysis. **, The size of the protein corresponding to the HCV p7 in GBV-B is 13 kDa; this protein could be cleaved into p7 and p6 proteins, of which the p7 protein, but not the p6 protein is critical for viability in vivo. Proposed names for GB viruses are in parentheses. on the relative translational efficiency of the IRES elements for GB viruses. The 59 NTRs of HCV isolates are approximately 340 nt long, whereas the 59 NTR of GBV- B is 445 nt, and the contained IRES directs the translation of a core protein. In contrast, the 59 NTRs for GBV-A and GBV-C/HGV are predicted to be longer based on in vitro translation studies that demonstrated that the AUG at position 556 of GBV-C/HGV was the codon that initiated translation (Simons et al., 1996). Sequence numbering is based on the infectious clone isolate (Xiang et al., 2000) (GenBank accession no. AF121950). Thus, GBV-A and some GBV-C/HGV isolates do not appear to encode a core protein (Kim & Fry, 1997; Leary et al., 1996b; Xiang et al., 1998). A signal peptidase cleavage site is predicted to occur 17 or 21 aa downstream of the putative initiation codon in GBV-C/HGV (Mohr & Stapleton, 2009), although it is doubtful that this small peptide could serve as the core (nucleocapsid) protein. Biophysical characterization of GBV-C/HGV particles, however, found that they appear to have a nucleocapsid (Xiang et al., 1998). Although there is limited experimental evidence, several potential hypo- theses have been put forward to explain the potential 238 source of the nucleocapsid protein. These include the possibility that the capsid forms from the very small cleaved peptide at the N terminus of the polyprotein (Xiang et al., 1998), or that a longer core protein is translated off an alternative reading frame on the genomic or negative strands of the GBV-C/HGV genome. Alternatively, the hypothesis that the virus utilizes a cellular protein to serve as the nucleocapsid protein has been raised (Theodore & Lemon, 1997). It is unclear which AUG codon initiates translation in GBV-D. Like GBV-C, there are multiple potential initiation codons in-frame with the GBV-D coding sequence. Specifically, there are five AUG codons between the 59 end of the GBV-D genome and nt 744 that are in-frame with the long ORF (GenBank accession nos GU566734 and GU566735). Of note, the predicted amino acid sequence of GBV-D starting at nt 744 is MAVLLLLSTGLAEG. The GBV-C predicted amino acid sequence starting at the AUG shown to initiate translation in vitro (Simons et al., 1996) is MAVLLLLLVVEAGA, thus sharing complete identity with the first seven amino acids of GBV-D. GBV-A and Journal of General Virology 92