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.

Importance: Although the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.

FIG 1

Expression of SARS-CoV nonstructural proteins. (A) Schematic of nsp3, nsp3N, nsp3C, nsp4, and nsp6 constructs used. UB1, ubiquitin-like domain 1; AC, acidic region; ADRP, ADP-ribose-1′′-phosphatase; SUD, SARS unique domain; UB2, ubiquitin-like domain 2; PLP2^PRO, papain-like protease; NAB, nucleic acid binding domain; G2M, group II-specific marker; TM, transmembrane region; ZF, putative metal-binding region; Y, Y region; h, HA epitope tag; b, biotinylation signal sequence; f, FLAG epitope tag. (B) Left panel: detection of nsp3 in SARS-CoV-infected cell lysate and nsp3-transfected cell lysate via anti-nsp3. Right panel: detection of nsp3 and nsp3N in transfected cell lysates via anti-nsp3. (C) Detection of nsp4, nsp6, nsp3N, and nsp3C in transfected cell lysates via anti-FLAG.

FIG 2

Intracellular localization of accumulation of SARS-CoV nonstructural proteins. (A) Upper panel: detection of nsp3 (green) and double-stranded RNA (dsRNA) (red) in SARS-CoV-infected HEK293T-ACE2 cells (MOI = 0.1, fixed 24 h postinfection [hpi]). Lower panel: detection of nsp3 (green) in nsp3-transfected HEK293T cells. (B) Detection of nsp3N (green), nsp3C (red), nsp4 (green), and nsp6 (green) in transfected HEK293T cells using anti-FLAG antibody. (C) Upper panel: detection of nsp3 (green) and nsp4 (red) in cotransfected HEK293T cells. Lower panel: detection of nsp3 (green) and nsp6 (red) in cotransfected HEK293T cells. (D) Time course experiment detecting nsp3 (green) in transfected cells (fixed at the indicated time points) over a 24-h period.

FIG 3

Disordered membrane body (DMB) and multilamellar and giant vesiculation (MGV) in SARS-CoV nsp3- and nsp3C-transfected cells. (A) DMB in nsp3-transfected cell. Zoomed region shows membrane detail. (B) MGV in nsp3-transfected cell. (C) DMB in nsp3C-transfected cell. Zoomed region shows membrane detail. (D) MGV in nsp3C-transfected cell.

FIG 4

Maze-like body (MLB) formation in SARS-CoV nsp3-nsp4-cotransfected cells. (A and B) Perinuclear localization and double-wall highlights (zoomed region). Interconnections to the endoplasmic reticulum (black arrowheads) and smooth-sided single membranes interrupting maze-like bodies (white arrowheads) are indicated.

FIG 5

Microtubule organizing center vesiculation (MTOCV) in SARS-CoV nsp6-transfected cells. (A) Untransfected control. (B) SARS-CoV nsp4-transfected cell. (C) SARS-CoV nsp6-transfected cell featuring MTOCV. (D) SARS-CoV nsp4-nsp6-cotransfected cell. Centrioles (black arrowheads) are indicated.

FIG 6

SARS-CoV-induced DMVs versus triple-transfection SARS-CoV nsp3-nsp4-nsp6-induced DMVs. (A and B) SARS-CoV-infected cells. MOI = 1, fixed 7 h postinfection. (C to F) nsp3-nsp4-nsp6-transfected cells. Clusters consisting of convoluted membrane tubules (white arrowheads) ending in double-membrane vesicles (black arrowheads) are indicated.