Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene.

摘要

Recombinant baculoviruses obtained by coinfection of insect cells with Autographa californica and Bombyx mori nuclear polyhedrosis viruses (AcNPV and BmNPV, respectively) possess a wider in vitro host range than either parent virus. To localize the DNA sequences responsible for this species specificity, we used a two-step method of production and selection of recombinant viruses with altered specificity. Sf9 cells, which are permissive for AcNPV, were first cotransfected with genomic AcNPV DNA and a complete or incomplete set of BmNPV restriction fragments. AcNPV-BmNPV recombinants from the Sf9 supernatant were then selected on the basis of ability to replicate in B. mori Bm5 cells, which are not permissive for AcNPV. Cotransfection of AcNPV DNA with the 7.6-kbp BmNPV Sma I-C fragment was sufficient to produce recombinants able to infect both Sf9 and Bm5 cells. A series of cotransfections with subclones of this fragment defined a 79-nt sequence within the p143 helicase gene capable of extending AcNPV host range in vitro. In this 79-nt region, BmNPV and AcNPV differ at six positions, corresponding to four amino acid substitutions. The involvement of the 79-nt region in species specificity control was confirmed by cotransfecting AcNPV DNA and gel-purified polymerase chain reaction products derived from the BmNPV p143 gene. Replacement in the AcNPV genome of three AcNPV-specific amino acids by the three corresponding BmNPV-specific amino acids at positions 556, 564, and 577 of the p143 protein extends AcNPV host range to B. mori larvae.