Using A Novel Endoplasmic Reticulum Fluorescent Marker Protein To Study The Morphological Changes Of The Endoplasmic Reticulum After Flavivirus Infectio

The endoplasmic reticulum(ER)is a highly complex and dynamic organelle that exhibits a variable morphology and closely communicates with other cellular organelles.It presents a complex and dense network structure in the cell,which poses a huge challenge to visualizing the ER ultrastructure.Traditional ER labeling methods,such as ER tracker staining and ER markers using fluorescent proteins(mCherry-KDEL and EGFP-Sec61β),have limitations such as weak fluorescence signals,low labeling efficiency,and easy photobleaching,making it difficult to visualize dynamic changes of the ER in living cells by superresolution microscopy.In this study,we designed a novel ER marker protein,RR-mNeonGreen,consisting of an N-terminal ER retention signal,a highly bright green fluorescent protein(mNeonGreen),and a C-terminal transmembrane domain.RR-mNeonGreen can colocalize with mCherry-KDEL,indicating that RR-mNeonGreen can effectively label the ER.When imaged by superresolution microscopy,ER labeled with RR-mNeonGreen exhibited higher structural resolution and better continuity of ER tubules.In addition,RR-mNeonGreen is resistant to photobleaching,allowing for long-term imaging of dynamic changes of the ER in living cells and tracking the interactions between the ER and mitochondria at high spatiotemporal resolution.As an important membranous organelle in eukaryotic cells,the ER plays an important role in the replication of many positive-stranded RNA viruses.Well-known flavivirus,such as Zika virus and dengue virus,and coronaviruses,such as SARS and SARS-CoV-2,are all positive-stranded RNA viruses,and their genome replication occurs in special membranous replication organelles.Both flaviviruses and coronaviruses hijack host cell ER and induce ER deformation to produce virus replication organelles.The formation mechanism and dynamic changes of these virus replication organelles have not been fully elucidated.Combined with RR-mNeonGreen and superresolution live-cell imaging technology,we investigated the structure and morphology of the ER induced by Zika virus infection.We found that Zika virus infection caused ER aggregation,forming ER clusters.Nonstructural proteins encoding by viral genome localized to these ER clusters,indicating that these ER clusters are virus replication organelles.Most notably,after Zika virus infection,the host ER converged to the prinuclear region as the main replication compartments(MRCs),suggesting that Zika virus may enhance its replication efficiency through this concentrated replication mode.In summary,we have constructed a novel ER marker(RR-mNeonGreen)that can efficiently and specifically label the ER.We indicated that RR-mNeonGreen is suitable for long-term superresolution live-cell imaging.Using this new ER marker,we investigated the structure and dynamic changes of the ER remodeled by Zika virus,revealing the mode of Zika virus replication in the MRCs at perinuclear regions.