Expression of Aleutian mink disease parvovirus proteins in a baculovirus vector system.
We have previously published a detailed transcription map of Aleutian mink disease parvovirus (ADV) and proposed a model for the translation of the two virion structural proteins (VP1 and VP2) and three nonstructural proteins (NS-1, NS-2, and NS-3) (S. Alexandersen, M. E. Bloom, and S. Perryman, J. Virol. 62:3684-3994, 1988). To verify and further characterize this model, we cloned the predicted open reading frames for NS-1, NS-2, NS-3, VP1-VP2, and VP2 alone into a recombinant baculovirus and expressed them in Sf9 insect cells. Expression of VP1-VP2 or VP2 alone in cDNA and in the genomic form was achieved. The expressed proteins had molecular weights similar to those of the corresponding proteins of wild-type ADV-G, although the ratio of VP1 to VP2 was altered. The recombinant baculovirus-expressed ADV VP1 and VP2 showed nuclear localization in Sf9 cells and were able to form particles indistinguishable, by electron microscopy, from wild-type virus. The large nonstructural protein, NS-1, showed predominantly nuclear localization in Sf9 cells when analyzed by immunofluorescence and had a molecular weight similar to that of wild-type ADV NS-1. Moreover, expression of NS-1 in Sf9 cells caused a change in morphology of the cells and resulted in 10-times-lower titers of recombinant baculovirus during infection, suggesting a cytostatic or cytotoxic action of this protein. The smaller NS-2 gene product seems to be located in the cytoplasm. When analyzed by Western immunoblotting, NS-2 comigrated with an approximately 16-kDa band seen in lysates of ADV-infected feline kidney cells. The putative NS-3 gene product exhibited a diffuse distribution in Sf9 cells and had a molecular weight of approximately 10,000. All of the expressed ADV-encoded proteins were recognized by sera from ADV-infected mink. Thus, expression of ADV cDNAs allowed assignment of the different mRNAs to the viral proteins observed during ADV infection in cell culture and supported our previously proposed ADV transcriptional and translational scheme. Moreover, the production of structural proteins from a full-length NS-2 mRNA may add to the repertoire of parvovirus gene expression.
Christensen
J
J
Department of Veterinary Microbiology, Royal Veterinary and Agricultural University of Copenhagen, Frederiksberg, Denmark.
Storgaard
T
T
Bloch
B
B
Alexandersen
S
S
Aasted
B
B
D000454:Aleutian Mink Disease Virus / Q000235:genetics*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D036022:Capsid Proteins; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D036022:Capsid Proteins; D011994:Recombinant Proteins; C442555:VP2 protein, Aleutian mink disease virus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins