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The Host Protein Reticulon 3.1A Is Utilized by Flaviviruses to Facilitate Membrane Remodelling

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The Host Protein Reticulon 3.1A Is Utilized by Flaviviruses to Facilitate Membrane Remodelling Article The Host Protein Reticulon 3.1A Is Utilized by Flaviviruses to Facilitate Membrane Remodelling Graphical Abstract Authors Turgut E. Aktepe, Susann Liebscher, Julia E. Prier, Cameron P. Simmons, Jason M. Mackenzie Correspondence [email protected] In Brief To study the underlying mechanism of flavivirus replication and membrane biogenesis, Aktepe et al. examine the role of the host membrane-shaping protein RTN3.1A during WNVKUN, DENV-2NGC, and ZIKVAFR replication. They find that RTN3.1A is required for NS4A-mediated membrane remodelling during biogenesis of the flavivirus replication complex. Highlights d Flavivirus sites of replication accumulate the host remodelling protein RTN3.1A d siRNA silencing of RTN3.1A impairs virus replication and membrane morphogenesis d RTN3.1A interacts with the WNVKUN NS4A protein to promote membrane remodelling d Flaviviruses utilize RTN3.1A differently during their replication cycles Aktepe et al., 2017, Cell Reports 21, 1639–1654 November 7, 2017 ª 2017 The Authors. https://doi.org/10.1016/j.celrep.2017.10.055 Cell Reports Article The Host Protein Reticulon 3.1A Is Utilized by Flaviviruses to Facilitate Membrane Remodelling Turgut E. Aktepe,1 Susann Liebscher,1 Julia E. Prier,1 Cameron P. Simmons,1 and Jason M. Mackenzie1,2,* 1Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia 2Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2017.10.055 SUMMARY et al., 1999; Miorin et al., 2013; Junjhon et al., 2014; Cortese et al., 2017). These membrane structures act as platforms Flaviviruses are enveloped, positive-sensed single- for efficient viral replication by compartmentalizing and hiding stranded RNA viruses that remodel host membranes, viral components from innate immune detection (Hoenen et al., incorporating both viral and host factors facilitating 2007; Overby et al., 2010; Hoenen et al., 2014); however, the viral replication. In this study, we identified a key exact mechanism underlying the formation of these structures role for the membrane-bending host protein Reticu- is not entirely understood (Mackenzie et al., 1999; Westaway lon 3.1 (RTN3.1A) during the replication cycle of three et al., 1997; Gillespie et al., 2010; Welsch et al., 2009). Viral replication is a complex process that requires many host flaviviruses: West Nile virus (WNV), Dengue virus factors at every stage of replication, including the formation (DENV), and Zika virus (ZIKV). We observed that, dur- of virally induced membrane structures (Ahlquist et al., 2003; ing infection, RTN3.1A is redistributed and recruited Wang and Li, 2012). It is now well established that the prolifera- to the viral replication complex, a recruitment facili- tion of intracellular membranes during WNV and DENV replica- tated via the WNV NS4A protein, however, not tion is mediated via the small hydrophobic protein NS4A, which, DENV or ZIKV NS4A. Critically, small interfering when individually expressed, is sufficient to proliferate mem- RNA (siRNA)-mediated knockdown of RTN3.1A branes similar to the CM/PC observed during viral replication expression attenuated WNV, DENV, and ZIKV repli- (Miller et al., 2007; Roosendaal et al., 2006). NS4A is a 16-kDa cation and severely affected the stability and abun- highly hydrophobic, ER-associated protein with three trans- dance of the NS4A protein, coinciding with a signifi- membrane (TM) regions, a membrane-associated region, and cant alternation and reduction of viral membrane an N-terminal cytoplasmic region (Mackenzie et al., 1998b; Miller et al., 2007). NS4A is also predicted to form part of the replication structures in the endoplasmic reticulum. These ob- complex (RC), determined from studies that have shown interac- servations identified a crucial role of RTN3.1A for tions with the replicative intermediates double-stranded RNA the viral remodelling of host membranes during effi- (dsRNA), NS1, NS2A, and NS5 (Mackenzie et al., 1998a) and cient flavivirus replication and the stabilization of via its interaction with the cellular scaffolding protein vimentin viral proteins within the endoplasmic reticulum. to aid RC formation (Teo and Chu, 2014). Additionally, mutations within the conserved amino acid and TM domains of NS4A INTRODUCTION impacted negatively on NS4A protein stability, cleavage effi- ciency, and oligomerization and duly on virus replication The Flaviviridae family contains numerous highly pathogenic (Ambrose and Mackenzie, 2011a, 2015; Stern et al., 2013; Lee viruses, such as West Nile virus (WNV), Dengue virus (DENV), et al., 2015). Zika virus (ZIKV), Japanese encephalitis virus (JEV), and Yellow The Reticulon (RTN) protein family is a large group of mem- Fever virus (YFV). To successfully replicate its small genome brane-associated proteins that are involved in vesicle formation size (11 kb), flaviviruses utilize both viral and host factors to and membrane morphogenesis. The RTN protein family consists aid in transcription, translation, immune evasion, and remodel- of four gene products (RTN1, RTN2, RTN3, and RTN4/Nogo), ling of the intracellular membranous environment (Romero- and, further, depending on promoters and alternative splicing, Brey and Bartenschlager, 2016). This remodelling of membranes it includes more than 300 proteins (Oertle et al., 2003; Moreira drives the biogenesis of characteristic flavivirus-induced mem- et al., 1999). A common feature of all RTN proteins is the C-ter- brane structures that are highly curved and complex, termed minal Reticulon Homology Domain (RHD) that consists of two vesicle packets (VPs), convoluted membrane (CM), and para- hairpin TM regions separated by a hydrophilic loop. The short to- crystalline arrays (PCs) (Westaway et al., 1997; Mackenzie pology of the hairpin TM sits on the outer leaflet of the ER mem- et al., 1996). Previous studies by multiple groups have identified brane and occupies a higher density of space, causing a positive that these membrane structures are derived from the endo- curvature of the membrane toward the cytoplasm (Yang and plasmic reticulum (ER) and also contain markers of the Golgi Strittmatter, 2007; Zurek et al., 2011). The RTN proteins localize apparatus (Welsch et al., 2009; Gillespie et al., 2010; Mackenzie to regions with high membrane curvature, and they are required Cell Reports 21, 1639–1654, November 7, 2017 ª 2017 The Authors. 1639 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). (legend on next page) 1640 Cell Reports 21, 1639–1654, November 7, 2017 to control the formation of ER tubules and increase peripheral ER analysis (Figure 1). Fixed samples were immune-labeled with an- sheets (Voeltz et al., 2006; Tolley et al., 2010; Westrate et al., tibodies to endogenous RTN3.1A and the viral markers dsRNA 2015). Previous studies have suggested a differential require- and the viral protein NS4A. Within uninfected cells (Figures 1A, ment of RTN proteins during the replication of +single-stranded I–IV, and 1B, I–IV), the RTN3.1A protein was observed to localize RNA (ssRNA) virus replication. Enterovirus 71 (EV71) requires as a reticular staining pattern in the cytoplasm and also confined RTN3A for replication as knockdown of RTN3A (via RNAi) in- to the perinuclear region. This distribution is consistent with pre- hibited EV71 replication (Tang et al., 2007). Additionally, it was vious observations indicating that RTN3.1A resides primarily shown that the EV71 2C protein directly interacts with RTN3A within the ER and Golgi (Kume et al., 2009). Upon infection to promote efficient replication (Tang et al., 2007). Similarly, the with WNVKUN, DENV-2NGC, or ZIKVAFR, dsRNA, which is a brome mosaic virus (BMV) 1a protein interacts with and incorpo- marker for viral replication complexes (Westaway et al., 1999), rates RTN proteins into the virus-induced spherules (comprising was observed to partially co-localize with RTN3.1A (Figures the RC), and deletion of a number of RTN proteins attenuated 1A, V–XVI). We observed the RTN3.1A staining as adjacent or BMV replication (Diaz et al., 2010). In contrast, RTN3A interacts juxtaposed to that of dsRNA (indicated by arrows), in addition with and negatively regulates Hepatitis C virus (HCV) NS4B to complete co-localization (indicated by arrowheads), and over- dimerization, and silencing of RTN3A enhanced HCV replication all with a more pronounced association of RTN3.1A with ZIKVAFR (Barajas et al., 2014). dsRNA compared to DENV-2NGC and WNVKUN (Figure 1C). Based on the observations described above, we aimed to Conversely, we observed significant co-localization between determine the role and impact of RTN3.1A on flavivirus replica- RTN3.1A and WNVKUN, DENV-2NGC, or ZIKVAFR NS4A during fla- tion, and we demonstrated that RTN3.1A is required for the effi- vivirus replication (Figures 1B, V–XVI, and 1D). Previous studies cient replication of WNV strain Kunjin (WNVKUN), DENV-2 strain on WNVKUN and DENV-2NGC have described that the NS4A pro- New Guinea-C (DENV-2NGC), and ZIKV African MR766 strain tein localizes primarily to the CM/PC structures (in addition to the (ZIKVAFR). Ultimately, we have demonstrated that flaviviruses uti- VP) (Mackenzie et al., 1998a; Welsch et al., 2009) and that NS4A lize the membrane-bending capacity of the viral NS4A protein has the intrinsic capability to remodel cytoplasmic membranes and the host RTN3.1A protein in concert to generate the
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