Evidence for the role of Rab11-positive recycling endosomes as intermediates in coronavirus egress from epithelial cells

Abstract

After their assembly by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)–Golgi interface, coronaviruses (CoVs) are released from their host cells following a pathway that remains poorly understood. The traditional view that CoV exit occurs via the constitutive secretory route has recently been questioned by studies suggesting that this process involves unconventional secretion. Here, using the avian infectious bronchitis virus (IBV) as a well-established model virus, we have applied confocal microscopy to investigate the pathway of CoV egress from epithelial Vero cells. We report a novel effect of IBV infection on cellular endomembranes, namely, the compaction of the pericentrosomal endocytic recycling compartment (ERC) defined by the GTPase Rab11, which coincides with the previously described Golgi fragmentation, as well as virus release. Despite Golgi disassembly, the IC elements containing the major IBV membrane protein (M)—which mostly associates with newly formed virus particles—maintain their close spatial connection with the Rab11-positive endocytic recycling system. Moreover, partial colocalization of the M protein with Rab11 was observed, whereas M displayed negligible overlap with LAMP-1, indicating that IBV egress does not occur via late endosomes or lysosomes. Synchronization of virus release using temperature-shift protocols was accompanied by increased colocalization of M and Rab11 in vesicular and vacuolar structures in the pericentrosomal region and at the cell periphery, most likely representing IBV-containing transport carriers. In conclusion, these results add CoVs to the growing list of viruses exploiting the endocytic recycling apparatus defined by Rab11 for their assembly and/or release.

Myoferlin is an essential component of late-stage vRNP trafficking vesicles for enveloped RNA viruses

“…Importantly, however, this relationship is conserved on RSV and SeV vRNA-trafficking vesicles. While the data discussed here directly present strong evidence for the importance of myoferlin for vRNA trafficking of IAV, RSV and SeV specifically, they imply that this would also be the case for all Rab11a-dependent viruses, including coronaviruses, 10 parainfluenza viruses, 9 Hantaviruses, 4 ebolaviruses 6 and flaviviruses. 5…”

“…While only three respiratory viruses that utilise Rab11a-mediated vRNP trafficking were investigated here, these data imply a conserved role for myoferlin in the replication of other Rab11-dependent viruses, including respiratory pathogens of the Coronaviridae and Paramyxoviridae families. 3,10,47…”

“…9 It has also been speculated that coronaviruses exploit this pathway during egress. 10 At late stages of infection of each virus, Rab11a-containing vesicles are exploited to traffic viral ribonucleoprotein (vRNP) complexes towards the apical surface so that virions are dispersed into the respiratory airways. 3,[11][12][13] A better understanding of this cellular trafficking system in both the presence and absence of infection could reveal important, novel insights broadly applicable to many virus families.…”

The role of lysosomes as intermediates in betacoronavirus PHEV egress from nerve cells

“…However, our findings and results of previous studies indicate that βCoVs, such as PHEV, SARS-CoV-2, and MHV, employ a lysosome-based egress pathway rather than a biosynthetic secretory egress pathway ( 6 ). Meanwhile, Saraste et al have reported evidence indicating that Rab11-positive recycling endosomes may function as transport carriers during the cellular exit of avian infectious bronchitis virus, a γCoV ( 24 ). Thus, conflicting findings concerning the roles of secretory compartments in CoV egress suggest that CoVs belonging to different genera may exit cells via different mechanisms.…”

In focus in Vienna: Microscopy and cellular organelles

“…Coronaviruses assembly occurs by their budding in the lumen of the intermediate compartment, a subcellular vesiculo-tubulo structure located at the endoplasmic reticulum–Golgi apparatus interface (Saraste et al 1987 ). The poorly understood release pathway of coronaviruses from their host cells was investigated by Saraste et al ( 2022 ) by employing confocal immunofluorescence microscopy. Virus infection resulted in the accumulation of recycling endosomes defined by the GTPase Rab11 next to the centrosome, which was intimately linked to virus-induced Golgi fragmentation and virus release.…”