EXTENSION OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS HOST-RANGE BY INTERSPECIFIC REPLACEMENT OF A SHORT DNA-SEQUENCE IN THE P143 HELICASE GENE

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 Sf 9 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 Sf 9 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.