Functional analyses of GB virus B p13 protein: development of a recombinant GB virus B hepatitis virus with a p7 protein.
GB virus B (GBV-B), which infects tamarins, is the virus most closely related to hepatitis C virus (HCV). HCV has a protein (p7) that is believed to form an ion channel. It is critical for viability. In vitro studies suggest that GBV-B has an analogous but larger protein (p13). We found that substitutions of the -1 and/or -3 residues of the putative cleavage sites (amino acid 613/614 and 732/733) abolished processing in vitro and rendered an infectious GBV-B clone nonviable in tamarins. Internal cleavage was predicted at two sites (amino acid 669/670 and 681/682), and in vitro analysis indicated processing at both sites, suggesting that p13 is processed into two components (p6 and p7). Mutants with substitution at amino acid 669 or 681 were viable in vivo, but the recovered viruses had changes at amino acid 669 and 681, respectively, which restored cleavage. A mutant lacking amino acid 614-681 (p6 plus part of p7) was nonviable. However, a mutant lacking amino acid 614-669 (p6) produced high titer viremia and acute resolving hepatitis; viruses recovered from both animals lacked the deleted sequence and had no other mutations. Thus, p6 was dispensable but p7 was essential for infectivity. The availability of a recombinant GBV-B virus containing a p7 protein with similarities to the HCV p7 will enhance the relevance of this model and will be of importance for identifying compounds that inhibit p7 function as additional therapeutic agents.
Takikawa
Shingo
S
Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Engle
Ronald E
RE
Emerson
Suzanne U
SU
Purcell
Robert H
RH
St Claire
Marisa
M
Bukh
Jens
J
D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D029406:GB virus B / Q000235:genetics* / Q000378:metabolism*; D005818:Genetic Engineering; D006801:Humans; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins