Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein

Regulatory elements interacting with DNA-binding proteins have been investigated in the promoter sequence of the early PE38 gene in the Autographa californica nuclear polyhedrosis virus (AcNPV). A GATA motif located 50 nucleotides upstream of the PE38 transcriptional start site is recognized differentially in the course of infection. As demonstrated by footprint and gel mobility shift assays, the GATA sequences TTATCT are protected by nuclear extracts from uninfected Spodoptera frugiperda cells and from S. frugiperda cells early postinfection (p.i.) but not by S. frugiperda cell extracts isolated 40 h p.i. We have compared the binding capacity of the insect GATA-like protein with that of the vertebrate GATA-1 factor identified as erythroid-specific factor. Our results indicate that a factor present in mouse erythroleukemia cells, presumably GATA-1, can bind to the insect GATA motif and vice versa. Evidence from transient expression studies suggests that the mutated GATA sequences do not influence PE38 promoter activity in cell culture.</jats:p

Identification of the very early transcribed baculovirus gene PE-38

We have started to identify early viral RNAs that are transcribed at 1 h after inoculation to investigate the mechanism involved in the regulation of early gene expression of Autographa californica nuclear polyhedrosis virus (AcNPV). Cloned viral DNA fragments were hybridized to Northern (RNA) blots of polyadenylated RNA isolated from Spodoptera frugiperda cells at 1, 2, and 6 h postinfection to localize very early transcripts. Subsequently we prepared a cDNA library of polyadenylated RNA transcribed at 1 h after inoculation to analyze the cDNA clones corresponding to the major early RNAs. We identified a gene located upstream of the immediate-early gene IE-N extending in the opposite direction. Because of the very early expression during AcNPV infection and the transient expression in uninfected cells, we conclude that we found an immediate-early gene, designated PE-38. The determination of the nucleotide sequence of PE-38 revealed one open reading frame potentially encoding a gene product of 38 kDa. Results of in vitro translation experiments suggest that a PE-38-specific polypeptide of approximately 38 kDa can be expressed. We have evidence from computer analyses that the predicted amino acid sequence includes two putative DNA-binding motifs, a zinc finger, and a leucine zipper.</jats:p

Expression of PE38 and IE2, viral members of the C3HC4 finger family, during baculovirus infection: PE38 and IE2 localize to distinct nuclear regions

The pe38 gene of Autographa californica nuclear polyhedrosis virus represents one of the major early transcripts after viral infection. The function of the pe38 protein, which contains a C3HC4 zinc finger motif, is still not understood. We have raised polyclonal antiserum against the pe38 protein, PE38, produced in bacteria to investigate pe38 expression in the course of infection. A approximately 38-kDa polypeptide is first detectable at 2 h postinfection and decreases rapidly after 24 h. During the late phases of infection, a smaller protein of approximately 20 kDa which cross-reacts with the PE38-specific antiserum is visible at a constant level until 120 h postinfection. Since the pe38 gene shares a divergent promoter unit with the ie2 gene (formerly IEN), we have compared the expressions of the two genes. Polyclonal antibodies were raised against the bacterially expressed ie2 protein. The temporal expression pattern of the approximately 49-kDa ie2 protein is comparable to that of the approximately 38-kDa pe38 protein. Furthermore, both proteins are present in the nuclear fraction of A. californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells, but the approximately 38-kDa pe38 protein is also detectable in the cytoplasm while the smaller protein of approximately 20 kDa is exclusively present in the cytoplasmic fraction. Immunofluorescence analysis reveals that PE38 and IE2 localize to distinct regions within the nucleus mainly detected after transfection of pe38- and ie2-expressing constructs.</jats:p