Transcription and promoter analysis of pif, an essential but low-expressed baculovirus gene

• Published in: Journal of general virology 2004-02, Vol.85 (2), p.331-341
• Main Authors: Gutierrez, S, Kikhno, I, Ferber, M.L
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blotting, Northern; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral; Larva - virology; Microbiology; Miscellaneous; Mutation; Nucleopolyhedroviruses - genetics; Nucleopolyhedroviruses - pathogenicity; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; RNA, Viral - analysis; Spodoptera - virology; Transcription, Genetic; Viral Structural Proteins - biosynthesis; Viral Structural Proteins - genetics; Virology; Virulence - genetics
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/vir.0.19623-0

Laboratoire de Pathologie Comparée, UR 1231 INRA, 30380 Saint Christol les Alès, France Correspondence Miguel López Ferber lopez{at}ensam.inra.fr The pif gene ( per os infectivity factor) of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) encodes a structural protein essential for oral infection. This protein is expressed in very low quantities. In this study, transcription and promoter analysis of SpliNPV pif were carried out to understand more fully the regulation of pif gene expression. Transcription in the pif gene region was examined using RT-PCR, Northern blot, primer extension, ribonuclease protection and 3' RACE. The pif gene was encoded by a late bicistronic messenger, which was characterized. This 1·9 kb messenger was present in very small amounts. In addition, this messenger was part of a set of six late mRNAs overlapping the pif sequence. A functional complementation assay was used to analyse the pif promoter. This assay allowed the detection of amounts of PIF which were sufficient for the production of orally infectious virions. The 13 bp region upstream from the initial ATG of pif was required and sufficient for the production of orally infectious virions. This promoter region was much shorter than the studied baculovirus promoters. A late promoter motif (TTAAG) is situated at the 5' end of this region. This motif was shown to be the promoter core by using single mutations of the motif in the complementation assay. These results suggest that the low expression of the pif gene is regulated chiefly at the transcriptional level. Present address: Department of Biochemical Genetics, Institute of Molecular Biology and Genetics, Ukrainian National Academy of Sciences, Zabolotny St 150, 03143 Kiev, Ukraine.