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Journal of Virology, January 2003, p. 535-545, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.535-545.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells{dagger}

Liqun Lu, Quansheng Du,{ddagger} and Nor Chejanovsky*

Entomology Department, Institute of Plant Protection, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel

Received 5 July 2002/ Accepted 24 September 2002

Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

* Corresponding author. Mailing address: Entomology Department, Institute of Plant Protection, The Volcani Center, POB 6, Bet Dagan 50250, Israel. Phone: (972) 3-968-3694. Fax: (972) 3-960-4180. E-mail: ninar{at}volcani.agri.gov.il.

{dagger} Contribution 517\01 from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.

{ddagger} Present address: Center for Cell Signaling, School of Medicine, University of Virginia, Charlottesville, VA 22908.

Journal of Virology, January 2003, p. 535-545, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.535-545.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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