Roles of the Endogenous Lunapark Protein during Flavivirus Replication

Abstract

The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.

Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection

“…3A ) were transfected into baby kidney hamster 21 (BHK-21) cells. The 3’UTR was engineered with an internal ribosome entry site (IRES) to drive the expression of a neomycin/kanamycin resistance gene IRES-NeoR/KanR, allowing for the selection of cells expressing the subgenomic replicons ( 28 ). We also characterized expression of the ESCRT proteins during siRNA treatment of BHK-21 cells.…”

“…The WNV Kunjin replicon was constructed based on the WNV Kunjin sequence (accession number AY274504), and the LGTV replicon was constructed based on the LGTV strain TP21 sequence (accession number NC003690). The constructs are driven by a T7 promoter for in vitro transcription to express the replicons as described in ( 28 ) ( Fig. 3A ).…”

Let’s Get Physical: Flavivirus-Host Protein–Protein Interactions in Replication and Pathogenesis

“…Similar to RTN and ATL, siRNA silencing of LNP results in significant reduction in flavivirus induced replication compartments and corresponding decreases in genome replication. Using Co-IP, Tran et al (2021) identified that TBEV NS4B interacted with LNP through its C-terminal region ( Tran et al, 2021 ). Additionally, ZIKV NS4A has been identified to interact with LNP, and may constitute virus-specific mechanisms of ER-remodeling ( Shah et al, 2018 ).…”

Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean–Congo Hemorrhagic Fever Virus

“…Protein electrophoresis and immunoblotting were conducted as previously described [24,38]. RIPA buffer (Thermoscientific, Waltham, MA, USA) with protease inhibitors (Sigma, St. Louis, MO, USA) was used to lyze cells for 20 min at 4 • C, followed by boiling in LDS sample buffer (Invitrogen).…”

“…Sera collected from each group were pooled and heat-inactivated by incubation at 56 • C for 30 min before being 5-fold serially diluted in DMEM. WNV KUN was rescued from infectious clones, as previously described [38], and was diluted so that there were approximately 50 infectious particles per volume. Next, the diluted sera were incubated for 1 h at 37 • C before infecting 90% confluent Vero cells growing on 24 well-plates for 1 h at 37 • C in 5% CO 2 .…”