<b>Targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ERGIC (endoplasmic reticulum Golgi intermediate compartment) and Golgi </b><b>complex </b>

Abstract

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) is a newly emerged phlebovirus identified in China, Japan, and South Korea. Phlebovirus glycoproteins (GP) play a key role in targeting viral structural components to the budding compartments in the ER-Golgi intermediate compartment (ERGIC) and Golgi complex. However, the role of SFTSV GP in targeting structural proteins to the ERGIC and Golgi complex remains unresolved. In this study, we show that SFTSV GP plays a significant role in targeting RNA-dependent RNA polymerase (L) and nucleocapsid protein (NP) to the budding sites. Confocal microscopy was used to investigate the subcellular localization of SFTSV structural proteins. In SFTSV-infected cells, GP and L localized to the ER, ERGIC and Golgi complex, whereas NP localized to the ERGIC and Golgi complex. In addition, GP colocalized with L and NP in infected cells. In cells singly transfected with GP, L or NP, GP localized to the same subcellular compartments as in infected cells. However, L or NP alone did not localize to the ER, ERGIC, or Golgi complex. Cotransfection experiments showed that GP altered the localization of L to the ERGIC and Golgi complex but not that of NP. Interestingly, plasmid-expressed NP fused with a hemagglutinin tag localized to the ERGIC and Golgi complex when expressed in SFTSV-infected cells and colocalised with GP, suggesting that GP plays a role in the subcellular localization of L and NP in infected cells. Thus, the SFTSV structural components start to assemble at the ERGIC to Golgi complex. GP is required for transporting L and NP to the ERGIC and Golgi complex. In addition, targeting of NP requires interaction with other factors besides GP.

A Model for the Production of Regulatory Grade Viral Hemorrhagic Fever Exposure Stocks: From Field Surveillance to Advanced Characterization of SFTSV

“…Viral GP was detected in the cytoplasm of infected cells and localized within the Golgi apparatus using SFTSV serum-specific pAb ( Figure 2 ). These data are consistent with previous reports of intracellular SFTSV localization [ 2 , 47 , 48 ].…”

“…Virus particles had an approximate diameter of 100 nm ( Figure 3 D), were seen adjacent to membrane structures identified as the Golgi apparatus, and were often found in vesicle-like structures ( Figure 3 B,C). These observations are consistent with previous studies [ 2 , 47 , 48 ] which demonstrate that SFTSV structural proteins are localized in the ERGIC compartment and Golgi complex of infected cells, suggesting that these viral components start to assemble in those cellular compartments and that the viral glycoproteins are required for transporting the RdRp and the NP proteins to the ERGIC and Golgi complex.…”

“…The virus was amplified and isolated in cell culture producing a high-titer viral stock, as determined by titration through a multiplex RT-qPCR-based TCID 50 assay. TEM analysis and immunofluorescence microscopy demonstrated clear virion-like particles and SFTSV properties consistent with previous studies [2,47,48].…”

Quantitative Proteomic Analysis Reveals Unfolded-Protein Response Involved in Severe Fever with Thrombocytopenia Syndrome Virus Infection

“…5). Activation of the UPR by SFTSV GP may be caused by accumulation of unfolded or misfolded proteins in the ER, since GP alone localizes in the ER, as well as in the ER-Golgi complex, and is responsible for recruiting RdRP and NP into these compartments during virus infection (48). Aggregated GP may modify membrane permeability of the ER, in turn altering ion homeostasis (10, 49).…”

Golgi Structure and Function in Health, Stress, and Diseases

“…As a highly dynamic organelle, Golgi serves as a membrane scaffold for multiple viruses, including infectious hepatitis C virus, enteroviruses, poliovirus, foot-and-mouth-disease virus, dengue virus, coronavirus, Kunjin virus, tick-borne encephalitis virus, rubella virus, and bunyamwera virus (Miller and Krijnse-Locker 2008;Harak and Lohmann 2015;Risco et al 2003;Salanueva et al 2003;Delgui et al 2013;Westerbeck and Machamer 2015), and is frequently fragmented after infection (Campadelli et al 1993;Salanueva et al 2003;Yadav et al 2016;Avitabile et al 1995;Lavi et al 1996;Hansen et al 2017;Rebmann et al 2016). Viruses use Golgi membranes directly and/or hijack master controllers of Golgi biogenesis and trafficking to generate vesicles that are used as the site of viral RNA replication (Quiner and Jackson 2010;Hansen et al 2017;Short et al 2013), wrapping (Sivan et al 2016;Alzhanova and Hruby 2007;Alzhanova and Hruby 2006;Nanbo et al 2018;Lundu et al 2018;Procter et al 2018), intracellular transduction (Nonnenmacher et al 2015), and secretion ). Viral infection triggers Golgi fragmentation via diverse mechanisms, ranging from phosphorylating key Golgi structural proteins such as GRASP65 (Rebmann et al 2016), activating the Src kinase to phosphorylate the Dynamin 2 GTPase (Martin et al 2017), targeting the immunity-related GTPase M (IRGM) to the Golgi to induce GBF1 phosphorylation (Hansen et al 2017), modulating vesicular trafficking (Yadav et al 2016;Johns et al 2014), to impeding the major histocompatibility complex (MHC) class I trafficking, antigen presentation, and/or cytokine secretion Rohde et al 2012).…”