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bioRxiv · 10.1101/2023.03.06.531455

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

Nagamine, T.·Sako, Y.
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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 forming 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 and that are dependent on the SLiM phosphorylation state. ImportanceSLiMs (short linear motifs) are compact non-globular protein interaction interfaces that mediate various cellular functions and as such are often mimicked by viruses to rewire cellular pathways. Here, we found that an unusual type of viral SLiM acts as a conformational switch rather than a cellular mimic. Prior to viral DNA replication, the baculovirus IE1 protein promotes early gene transcription within its focus. During viral DNA replication, the IE1 foci expand and fuse to form the virogenic stroma where late virus replication events occur. Our results indicate that IE1 undergoes a conformational change that is dependent on the infection stage and SLiM phosphorylation. This work provides new insights into the role SLiMs play in heterotypic and homotypic protein-protein interactions.

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Nagamine, T., Sako, Y.. 2023-03-07. A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability. https://doi.org/10.1101/2023.03.06.531455

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