Expression of snowshoe hare bunyavirus S RNA coding proteins by recombinant baculoviruses.
Recombinant baculoviruses have been constructed that express the two snowshoe hare (SSH) bunyavirus proteins coded in overlapping reading frames of the SSH S viral-complementary RNA species (namely the nucleoprotein, N, and the nonstructural protein, NSS). The 26.5 kDa N protein, which is read from the first AUG of the mRNA containing the SSH S sequence, was expressed at a high level (estimated to be ca 40% of the stained cellular proteins in recombinant baculovirus-infected Spodoptera frugiperda cells). This level of expression was much higher than that of the 10.5 kDa NSS protein made at the same time (estimated to be less than 1% of the stained proteins), presumably due in part to lower levels of translation initiation from the second AUG (19 nucleotides downstream). Bal31 nuclease digestion was used to delete the first ATG of the SSH DNA sequence in the baculovirus transfer vector and BamHI was used to remove downstream N coding sequences. A second recombinant baculovirus was constructed from the products that only expressed the SSH NSS protein. The yield of NSS protein was estimated to be of the order of ca 2% of the stained cellular proteins. A third recombinant transfer vector made from the products of the Bal31 digestion, fortuitously possessed a new ATG 8-10 nucleotides upstream of the NSS ATG. A recombinant virus derived from this vector synthesized essentially similar quantities (ca 2% each) of both the NSS protein and a 16.7 kDa N-related product.
Urakawa
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T
NERC Institute of Virology, Oxford, U.K.
Small
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DA
Bishop
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DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002043:Bunyaviridae / Q000235:genetics*; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D003062:Codon; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D004247:DNA