A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability.

Abstract

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.