Identification and characterization of the IE-N gene from the Autographa californica Nuclear Polyhedrosis Virus

Abstract

To study temporal regulation of Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genes, the delayed early 39K gene was cloned from AcMNPV and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Co-transfection with viral DNA digested with BglII suggested that a viral element was involved in the activation of the delayed early 39K promoter. This BglII-sensitive element, was localized to the PstI-N fragment of AcMNPV DNA. A 1330 nucleotide RNA spanning the BglII site was transcribed when the PstI-N fragment was transiently expressed in S. frugiperda cells. This gene encoded by the PstI-N fragment was designated IE-N. The nucleotide sequence of the IE-N gene was determined and shown to encode a serine- and glutamine-rich protein with a predicted molecular weight of 47,000. A polypeptide produced by the IE-N coding region can be detected by in vitro translation, radio-immunoprecipitation detection of an IE-NCAT fusion protein, and by protein activity. IE-N mRNA was abundantly expressed early, but not late, after infection with AcMNPV. In addition, IE-N mRNA and activity can be detected in S. frugiperda cells transiently expressing the PstI-N fragment. Because IE-N did not require the synthesis of AcMNPV gene products to be expressed, IE-N is an immediate early gene. Three viral factors regulated the transient expression of IE-N. These factors were the viral enhancer hr1, immediate early gene IE-1, and the gene product of IE-N. The hr1 stimulated expression when linked in cis to the IE-N gene. This enhancement was independent of position and orientation with respect to the IE-N gene. Co-expression of IE-1 with IE-N reduced the overall expression of IE-N. This decrease in IE-N expression by IE-1 was mediated by the hr1 enhancer sequences linked to the IE-N gene. The IE-N gene product increased expression from the IE-N gene. This stimulation was dependent upon the promoter of IE-N. In addition to increasing its own expression and that of the delayed early gene 39K, IE-N also stimulated expression of the immediate early IE-1 gene. IE-N, therefore, appears to produce a protein which functions in activating AcMNPV immediate early gene expression and possibly delayed early gene expression, as well.