CELLULAR AND MOLECULAR PATHWAYS CONTROLLING SARS-COV2 ASSEMBLY AND EGRESS

SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus belonging to the family of Coronaviridae. Coronaviruses assemble on intracellular membranes, differently from many other RNA viruses that assemble and egress directly at the plasma membrane. This feature adds an additional layer of complexity to the study of the coronavirus life cycle as, once assembled, virions have to traffic through host intracellular membranes to reach the plasma membrane to be released. The assembly of SARS-CoV-2 virions occurs at the ER-Golgi intermediate compartment (ERGIC), also named vesicular-tubular cluster (VTC). Here, the structural proteins Spike (S), Membrane (M) and Envelope (E), after being translocated from the endoplasmic reticulum (ER) to the ERGIC, interact with the complex formed by viral RNA and the Nucleocapsid (N) protein (ribonucleocapsid) on the cytosolic side of the membrane and form the viral particle by budding into the lumen of the ERGIC. From here, newly formed virions would be expected to enter the Golgi complex and reach the plasma membrane to egress but the intermediate stations traversed by the virus and the molecular machineries that host cells put in place to counteract viral propagation have not been clearly established. Many genetic screens have been performed to find host factors involved in SARS-CoV-2 infection. We filtered the results of these screens to select the host proteins involved in membrane trafficking and focused our attention on the Endosomal Complex Required for Transport (ESCRT) machinery that appears to behave as an anti-viral factor. To address the mechanism by which this molecular machinery acts as a restriction factor, we first used a reductionist approach based on transfection of single viral structural proteins, and then progressed to viral infection experiments. We found that the SARS-CoV-2 E protein is able to recruit the ESCRT complex via syntenin and Alix, thus defining the chain of molecular interactions that leads to the binding of this complex by a viral protein. Using immunofluorescence analysis, we found that this interaction occurs at late endosomes, where the ESCRT complex mediates the budding of the E protein into the lumen of the inner vesicles of multivesicular bodies. To study the role of ESCRT in viral infection, we took advantage of a cell line in which the ESCRT function can be impaired in an inducible manner through the expression of the dominant negative mutant of VPS4, the ATPase responsible for ESCRT disassembly. We found an increase in viral spread upon ESCRT machinery impairment, thus confirming the anti-viral role of this complex. Importantly, we found that viral structural proteins are degraded in lysosomes in infected cells, and also in transfected cells but only when the E protein is exogenously expressed. Our evidence led us to hypothesize that the ESCRT complex, through the interaction with the E protein, could be responsible for the degradation of single structural proteins and/or intact virions inside lysosomes. We then addressed the role of the secretory pathway in SARS-CoV-2 egress, which has been recently questioned. We found that the release of infectious viral particles is strongly impaired by Brefeldin A, a fungal toxin which inhibits anterograde transport, without affecting viral replication. Our data point to a pivotal role of the Golgi complex and secretory pathway for the release of viral particles and we found the ESCRT machinery as a restriction factor for viral infection.

Grantor dc:publisher

Università degli Studi di Milano

Year dc:date

2024

Author dc:creator

• SERGIO, MARIA CONCETTA

Contributors dc:contributor

• supervisore: M. A. De Matteis ; phd coordinator: S. Minucci
• M.C. Sergio
• MINUCCI, SAVERIO

• SARS-CoV-2
• membrane trafficking
• ESCRT
• egress routes
• Settore BIO/13 - Biologia Applicata

Language dc:language

eng

Handle dc:identifier

https://hdl.handle.net/2434/1046330

OAI identifier oai:identifier

oai:air.unimi.it:2434/1046330