Bluetongue virus tubules made in insect cells by recombinant baculoviruses: expression of the NS1 gene of bluetongue virus serotype 10

Abstract

Bluetongue virus (BTV) forms tubules in mammalian cells. These tubules appear to be composed of only one type of protein, NS1, a major nonstructural protein of the virus. To obtain direct evidence for the origin of the tubules, the complete M6 gene of BTV serotype 10 was inserted into the baculovirus transfer vector pAcYM1, so that it was under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. After cotransfection of Spodoptera frugiperda cells with wild-type A. californica nuclear polyhedrosis virus DNA in the presence of recombinant transfer vector DNA, polyhedrin-negative baculoviruses were recovered. When S. frugiperda cells were infected with one of the derived recombinant viruses, a protein similar in size and antigenic properties to the authentic BTV NS1 protein was made (representing ca. 50% of the stained cellular proteins). The protein reacted with BTV antibody and formed numerous tubular structures in the cytoplasm of S. frugiperda cells. The tubular structures have been purified to homogeneity from infected-cell extracts by gradient centrifugation. By enzyme-linked immunosorbent assay, the recombinant virus antigen has been used to identify antibodies to five United States BTV serotypes in infected sheep sera, indicating the potentiality of the expressed protein as a group-reactive antigen in the diagnosis of BTV infections.

Validation of a reverse transcriptase multiplex PCR test for the serotype determination of U.S. isolates of bluetongue virus

“…Four nonstructural proteins have been identified in BTV-infected cells (Van Dijk and Huismans, 1988). NSl has been associated with the formation of tubules in BTV-infected cells (Urakawa and Roy, 1988). The tubules, which are characteristic of BTV-infected cells, are found throughout the cytoplasm.…”

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells

“…For construction of recombinant transfer vectors, various BTV genes were selected. Each BTV DNA fragment was excised by digesting with BamHI enzyme from the single transfer vectors described previously (15)(16)(17)(18)(19). Each firgment was directly cloned into the BamHI or Bgfll sites of the triple or quadruple vectors.…”

“…When BTV VP6, or VP7 or the non-structural NS1 protein was expressed by single gene recombinant baculoviruses, all three proteins were synthesized in abundance, reaching up to 50 to 70% of the total protein mass of the infected S.frugiperda cells (16,17,19). Therefore, the genes encoding these proteins were chosen for insertion into the triple gene transfer vector.…”

“…To express four proteins in the quadruple vector we selected the genes that were either expressed at a high level in insect cells (eg, VP2, VP6, VP7, NS1) by single expression vector systems or can be assembled into a morphological structure in a native configuration (10,16,17,19). For high level expression VP2, VP6, VP7 and NSl were isolated from the corresponding single gene recombinant expression vector and cloned into pAcAB4 and the transfer vector pAcAB4.VP2/VP6/NS1/VP7 was constructed.…”

Characterization and modification of the carboxy-terminal sequences of bluetongue virus type 10 NS1 protein in relation to tubule formation and location of an antigenic epitope in the vicinity of the carboxy terminus of the protein

“…A cDNA representing BTV-10 M6 dsRNA has been cloned. Expression of the encoded NS1 protein by recombinant baculoviruses results in the synthesis of tubules in insect cells, proving that tubule formation requires no other BTV gene product (27). The expressed tubules have been purified, and their biochemical and biophysical properties have been analyzed.…”