Processing of dengue virus type 2 structural proteins containing deletions in hydrophobic domains.
The 5' end of the genome of the dengue virus type 2 encoding the structural proteins was expressed using recombinant vaccinia virus. Three additional recombinants derived by deletion of selected dengue sequences within the parental construct were also expressed. They were designed to assess the role of hydrophobic domains in the processing of the viral polyprotein in intact cells. The first construct contained a deletion of nucleotides encoding most of the C protein; nucleotides encoding the hydrophobic domain at the carboxy terminus were retained. The second and third constructs contained smaller deletions of 72 bp and 129 bp encoding hydrophobic domains at the carboxy termini of C and prM respectively. Indirect immunofluorescence and radioimmunoprecipitation were used to detect prM and E in cells infected with recombinant viruses. The results showed that deletion of 90% of C had no apparent effect on the processing of prM and E, and that the signal sequence for E at the carboxy terminus of prM was active in the absence of the upstream signal sequence for prM at the carboxy terminus of C. Deletion of the hydrophobic sequences preceding the amino terminus of E prevented cleavage at the prM-E junction. These results obtained using infected cells were consistent with the published findings for the translation of flavivirus RNA in vitro, and indicated the importance of membrane association in the cleavage of structural proteins from the flavivirus polyprotein. In addition, cells infected with the recombinant virus containing the large deletion in the C coding region released the E glycoprotein into the culture medium.
Gruenberg
A
A
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Wright
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral; D003716:Dengue Virus / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D015531:Radioimmunoprecipitation Assay; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000378:metabolism
D004279:DNA, Viral; C051821:E-glycoprotein, Dengue virus type 2; D021382:Protein Sorting Signals; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins