Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of nonstructural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells

Virus-specific tubules are characteristic of orbivirus infections and are likely to play an important role in virus morphogenesis. It has been shown that for bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae, the virus-encoded NS1 protein forms tubules in insect cells when the BTV segment M6 gene is expressed by using a baculovirus vector. To understand the function of NS1 tubules and to identify the sequences involved in their polymerization, a series of mutant NS1 genes was generated and expressed in insect cell cultures by using baculovirus vectors. Three of the mutants were deletion mutants. One (AcNS1.dNT10) lacked 10 of the amino-terminal amino acids, and the other two mutants (AcNS1.dCT20 and AcNS1.dCT43) lacked 20 or 43 of the carboxy-terminal amino acids. In addition, site-directed mutants were constructed in which various single cysteines or pairs of cysteines were changed to serines. The ability of each mutant protein to form tubules was investigated. None of the deletion mutants formed tubules. The constructs in which the cysteines at amino acid positions 337 and/or 340 were replaced by serines (e.g., AcNS1.C337S,C340S) also did not form tubules. Instead, the NS1 protein of these and the deletion mutants made ribbon-like structures which formed large aggregates. Mutations involving six other cysteines (i.e., AcNS1.C37S,C43S,AcNS1.C462S,C465S, AcNS1.C104S, and AcNS1.C364S) produced tubules. The results show that both the amino and carboxy termini of the NS1 protein molecule and the cysteines at residues 337 and 340 are essential for tubule formation.