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Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression.

ABSTRACT: An Autographa californica nuclear polyhedrosis virus (AcMNPV) gene required in transient expression assays for late and very late viral gene expression was identified, sequenced, and transcriptionally mapped. This gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the AcMNPV physical map. It was identified by cotransfecting Spodoptera frugiperda cultured cells with a collection of overlapping cloned DNA fragments covering the entire AcMNPV genome and a reporter gene controlled by an early, late, or very late AcMNPV promoter. Omission of the DNA fragment containing lef-1 curtailed most late and very late gene expression but not early gene expression. lef-1 was found to be an early gene transcribed as a 1.8-kb RNA in the presence of the protein synthesis inhibitor cycloheximide. The C terminus of the predicted polypeptide product, LEF-1, contained a sequence motif characteristic of nucleoside triphosphate-binding sites.

SUBMITTER: Passarelli AL

PROVIDER: S-EPMC237694 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression.

Passarelli A L AL Miller L K LK

Journal of virology 19930601 6

An Autographa californica nuclear polyhedrosis virus (AcMNPV) gene required in transient expression assays for late and very late viral gene expression was identified, sequenced, and transcriptionally mapped. This gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the AcMNPV physical map. It was identified by cotransfecting Spodoptera frugiperda cultured cells with a collection of overlapping cloned DNA fragments covering the entire AcMNPV genome and ...[more]

PMID: 8497062

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Project description:UnlabelledMany viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein, P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited similar distribution patterns in infected cells, and coimmunoprecipitation confirmed the interaction between them. Upon pk1 deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes was downregulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished from Sf9 cells transfected with the pk1 deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1 dependent. Replacement of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of the pk1 deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insights into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics.ImportanceDiverse posttranslational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to regulate gene expression. Many viruses also utilize host- or virus-induced chromatin machinery to promote efficient infections. Baculoviruses encode a protamine-like protein, P6.9, which is required for a variety of processes in the infection cycle. Currently, P6.9's PTM sites and its regulating factors remain unknown. Here, we found that P6.9 could be categorized as unphosphorylated, hypophosphorylated, and hyperphosphorylated species and that a virus-encoded serine/threonine kinase, PK1, was essential for P6.9 hyperphosphorylation. Abundant PTM sites on P6.9 were identified, among which 7 Ser/Thr phosphorylated sites were PK1 dependent. Mutation of these Ser/Thr sites reduced very late viral gene transcription and viral infectivity, indicating that the PK1-mediated P6.9 hyperphosphorylation contributes to viral proliferation. These data suggest that a code exists in the sophisticated PTM of viral protamine-like proteins and participates in viral gene transcription.

Dataset Information

Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression.

Publications

Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression.

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