Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles

Abstract

Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Furthermore, RdRp or nascent viral RNA has not been detected inside DMVs, and ultrastructural analysis could not confirm any connection between the DMV interior and the cytoplasm (38), raising questions about the import and export of ribonucleotide precursors and produced RNAs exported from RNA synthesis areas (44). The coexpression of the SARS-CoV transmembrane nonstructural proteins nsp3, nsp4, and nsp6 resulted in the formation of CMs and DMVs (45), suggesting a function in the biogenesis of the membranous replicative structures, and also in the anchoring of the RTC (46–48). …”

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles

“…Coronaviruses form a double-membrane organelle (DMO) derived from the endoplasmic reticulum (ER) that contains viral RNA and replicase proteins (Deming et al, 2007;Hagemeijer et al, 2010;Oostra et al, 2007;Shi et al, 1999;van der Meer et al, 1999). In the DMO, two lipid bilayers are held at a constant distance of about 20 nm (Angelini et al, 2013). Electron microscopy and tomography studies have revealed that the replicative organelles of coronaviruses and the related arteriviruses are drawn from a repertoire of paired-membranes, including open-ended spherules, closed double-membrane vesicles, and both planar and convoluted paired membranes (Knoops et al, 2008(Knoops et al, , 2012Maier et al, 2013).…”

Competitive Fitness in Coronaviruses Is Not Correlated with Size or Number of Double-Membrane Vesicles under Reduced-Temperature Growth Conditions

“…One randomly oriented ultrathin section per cell was examined in order to compare the sizes and numbers of intracellular organelles virions, as described previously (17). …”

“…The conserved gene cluster from the nsp3 gene to the nsp6 gene contains all the factors necessary for DMV formation: nsp3, nsp4, and nsp6 are needed to form DMV clusters (17) after essential processing by proteinases located in nsp3 (18) and nsp5 (19). Electron microscopy suggests that nsp3 induces membrane proliferation (17).…”