Synthesis of bluetongue virus chimeric VP3 molecules and their interactions with VP7 protein to assemble into virus core-like particles.
Bluetongue virus (BTV) core-like particles (CLPs) are formed in the cytoplasm of insect cells when only two major proteins (VP3 and VP7) of the BTV core are expressed by baculovirus vectors (T. J. French and P. Roy, 1990, J. Virol. 64, 1530-1536). We have recently reported that five small internal deletion mutants of VP3 form CLPs when provided with unmodified VP7 protein (D1-5; S. Tanaka and P. Roy, 1994, J. Virol. 68, 2795-2802). To investigate whether foreign sequences can be inserted into VP3 and to determine their effect on CLP formation, three of these internal regions (D1, D2, and D5), as well as the carboxy terminus, were modified to create unique restriction enzyme sites, thereby replacing VP3 coding regions with shorter synthetic sequences. Each modified VP3 gene was used to generate baculovirus expression vectors (D1I, D2I, D5I, and VP3C, respectively). Other than the D5I mutant, the mutants formed CLPs when expressed in the presence of VP7. Subsequently, T7 tag epitopes were inserted into the D1I, D2I, and VP3C restriction sites and recombinant baculoviruses were generated to express chimeric VP3 proteins (VP3D1IT, VP3D2IT, and VP3CT). Each chimeric protein retained the ability to form CLPs when VP7 protein was provided. In another construction an immunogenic sequence representing a bovine leukemia virus (BLV) glycoprotein peptide was incorporated into the carboxy terminus of VP3 and the derived CLPs were used to raise antibodies that reacted with the BLV antigen. The results suggest that the VP3 molecule can accommodate foreign sequences at certain sites without perturbing their ability to form CLPs with VP7.
Tanaka
S
S
Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Mikhailov
M
M
Roy
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001820:Bluetongue virus / Q000235:genetics / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000166:cytology; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
D001427:Bacterial Toxins; D004279:DNA, Viral; D004768:Enterotoxins; D000939:Epitopes; D011993:Recombinant Fusion Proteins; C074954:VP3 protein, Bluetongue virus; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C041348:tcdA protein, Clostridium difficile; C066862:VP7 protein, orbivirus