Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively

Abstract

The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.

Post-translational regulation and modifications of flavivirus structural proteins

“…Although this theory has been supported by immunofluorescence assays (Lorenz et al, 2003;Mackenzie & Westaway, 2001), the exact conformation of the prM/E heterodimers (i.e. trimeric spikes, as in immature virions) at this site has yet to be ascertained.…”

“…Although this is the classical model of flavivirus assembly, it may not be universal, as at least one report concerning WNV-Sarafend has demonstrated unusual virion budding at the plasma membrane (Ng et al, 1994). Although visualization of these discrete events on the ER is often limited by their rapid kinetics, co-localization experiments with ER markers have demonstrated prM and E residing at this organelle by immunofluorescence microscopy (Lorenz et al, 2003;Mackenzie & Westaway, 2001) and virion accumulation in ER cisternae by immunogold electron microscopy (Mackenzie & Westaway, 2001;Welsch et al, 2009). Recent investigations utilizing electron tomography have even purported to demonstrate direct visualization of flavivirus budding into the ER lumen at membrane sites directly opposite the pores of VPs (Junjhon et al, 2014;Welsch et al, 2009).…”

“…4). Virions that have accumulated in the ER cisternae are transported to the Golgi apparatus in individual vesicles for glycan maturation (Mackenzie & Westaway, 2001; Welsch et al, 2009). Transition from the ER through to the trans-Golgi compartment involves a drop in pH from 7.2 to 6.0, which induces a structural rearrangement from 60 prM/E heterotrimeric spikes to 90 prM/E heterodimers arranged in a herringbone pattern that is flattened parallel to the virion Zhang et al, 2003), a process that is reversible prior to furin cleavage (Fig.…”

“…Flavivirus prM also contains two C-terminal transmembrane helices anchoring the protein to the ER membrane, the second of which acts as a signal sequence for luminal translocation of the E protein (Hsieh et al, 2011). Nascent prM is initially glycosylated in the ER lumen at between one and three sites within the pr portion of the protein, with glycan maturation occurring during transit through the Golgi apparatus (Mackenzie & Westaway, 2001). …”

Identification of a dominant endoplasmic reticulum-retention signal in yellow fever virus pre-membrane protein

“…The flavivirus envelope protein heterodimer prME plays a major role in the budding process leading to the formation of the virus particle (reviewed by Heinz & Allison, 2000). After budding in the ER, the virus particles are transported through the normal secretory pathway (Lorenz et al, 2003;Mackenzie & Westaway, 2001). The presence of several ERretention signals is important for the ER localization of prME, a major player of the budding process.…”