The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle

Abstract

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication 1 . The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors 2 , but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents. SARS-CoV-2 extensively rearranges host cellular membranes into replication organelles that provide a microenvironment conducive to RNA synthesis and protection from host sensor and defence systems 1,2 . The 16 viral NSPs that are released from polyproteins pp1a and pp1ab by 2 viral proteases include 13 cytosolic proteins, which are involved in RNA replication, and 3 transmembrane proteins, NSP3, NSP4 and NSP6. Studies on other coronaviruses suggest that NSP3 and NSP4, with a hitherto undefined contribution from NSP6, are responsible for generating the replication organelles 3-6 . Despite considerable advances in our understanding of the ultrastructure of the SARS-CoV-2 replication organelle 2,7,8 , mechanistic insights into its biogenesis have so far been limited. In particular, there is at present-to our knowledge-no information on the role of NSP6 in this process. Of note, six SARS-CoV-2 'variants of concern' (VOCs) (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Eta (B.1.525), Iota (B.1.526) 9 and Lambda (C.37) 10 ) share a three-amino-acid deletion in NSP6 (NSP6(ΔSGF)), in addition to the more noted mutations in the spike protein; this finding adds further impetus to the need to examine the role of NSP6 in the biogenesis of replication organelles and in the replication of SARS-CoV-2. NSP6 induces ER zipperingWe tagged SARS-CoV-2 NSP6 at either the N or the C terminus. C-terminally tagged NSP6 showed a diffuse distribution in the ER (Fig. 1a and Extended Data Fig. ...

Atlas of interactions between SARS-CoV-2 macromolecules and host proteins

“…We found that the interactome of NSP4, NSP6, M, ORF7A, and ORF7B significantly enriched the term of nuclear outer membrane-ER membrane network ( Fig. 3 C), consistent with that M protein was a well-known membrane protein and recent findings showing that NSP4 and NSP6 were membrane proteins of double-membrane vesicles (DMVs) ( Ricciardi et al, 2022 ). The results suggest that ORF7A and ORF7B were most likely proteins preferred to localize at the membrane, especially ORF7A containing a potential transmembrane domain ( Samavarchi-Tehrani et al, 2020 ).…”

Some natural compounds and their analogues having potent anti- SARS-CoV-2 and anti-proteases activities as lead molecules in drug discovery for COVID-19

“…Six variants of SARS-CoV-2, namely Alpha, Beta, Gamma, Eta, Iota and Lambda have a three aminoacid residues deletion event (ΔSGF, in positions 106–108) during evolution, in the predicted second and longest nsp6 luminal loop, and have higher zippering activity, which in turn increases transmissibility, infectivity and immune escape of the virus. Moreover, viral nsp6 acts as an organizer of DMV clusters and mediates a contact with lipid droplets (LDs) through LD-tethering complex DFCP1-RAB18 [ 36 ]. Available evidence demonstrates that the RO and the LD contacts are important players in the development and progression of viral infection via utilization of released fatty acids from LD generated by lipophagy, as an energy source for morphogenesis of progeny virions [ 37 ].…”

The Gene Sequence of the Proteins of SARS-CoV-2

“…The nsp6 is transmembrane protein (Thomas, 2021) and act as an organizer of DMV clusters mediated through lipid droplet-derived lipids. The properly formed nsp6 connectors and lipid droplets are required for the replication of SARS-CoV-2 (Ricciardi et al 2022). Nsp6 also induce in ammatory cell death in lung epithelial cells (Sun et al 2022).…”