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. 2017 Nov 21;8(6):e01658-17.
doi: 10.1128/mBio.01658-17.

Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication

Diede Oudshoorn  1 , Kevin Rijs  1 , Ronald W A L Limpens  2 , Kevin Groen  1 , Abraham J Koster  2 , Eric J Snijder  1 , Marjolein Kikkert  3 , Montserrat Bárcena  4
Affiliations

Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication

Diede Oudshoorn et al. mBio. .

Abstract

Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp's) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.IMPORTANCE The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp's) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation.

Keywords: convoluted membranes; electron tomography; membrane structure; nidoviruses; nonstructural proteins; replication complex; replication organelle biogenesis; replication structures; viral factory; viral protein.

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Figures

FIG 1
FIG 1
MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PLpro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty pCAGGS vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with anti-SARS-CoV nsp3 serum that cross-reacts with MERS-CoV nsp3 (21), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [35S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
FIG 2
FIG 2
MERS-CoV nsp3 and nsp4 induce modification of intracellular membranes. (A and B) HuH-7 cells were infected with MERS-CoV (A) or mock infected (B) and analyzed at 10 h p.i. using EM. Several DMVs are indicated with red asterisks, and several spherules are indicated with red arrows. (C to F) HuH-7 cells were transfected with constructs expressing either individual nsp’s (C and D) or both nsp3 and nsp4, following either cotransfection with two plasmids (nsp3 + nsp4) or expression of a self-cleaving precursor (nsp3-4) (E and F), and analyzed using EM at 24 h posttransfection. (E and F) Some stretches of zippered ER are indicated with red arrows, and several DMVs are indicated with red asterisks. N, nucleus; G, Golgi apparatus; M, mitochondria; LD, lipid droplet; CM, convoluted membranes; DMB, disordered-membrane body; MSM, clusters of modified single membranes. Bars, 500 nm.
FIG 3
FIG 3
MERS-CoV nsp3 and nsp4 induce the formation of DMVs that are organized in an RVN. HuH-7 cells were cotransfected with constructs expressing nsp3 and nsp4 or the nsp3-4 precursor and fixed for ET analysis. (A) Overviews of reconstructed tomograms (available as Movies S1 and S2, respectively) for both conditions. Some of the fully reconstructed closed DMVs are indicated with red asterisks. (B) Zippered ER curving into putative intermediates during DMV biogenesis (indicated with red arrows) is shown. Two DMVs that are enclosed within other DMVs are indicated with red asterisks. (C) Examples of connections between DMVs and (zippered) ER (indicated with red arrows). All the images are virtual 5-nm-thick slices from the reconstructed tomograms. Bars, 250 nm.
FIG 4
FIG 4
Coexpression of MERS-CoV nsp6 does not alter DMV morphology. (A) Scaled schematic overview of MERS-CoV nsp3-6 constructs. Amino acid numbers at the top are the positions in MERS-CoV pp1a. Expected sites of cleavage by PLpro and Mpro are indicated. TM, transmembrane domain. Epitope tags used are indicated with ovals. (B) 293T cells were transfected with indicated plasmids and metabolically labeled with [35S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with indicated antibodies, separated on an SDS-PAGE gel, and imaged using a phosphorimager. The ~130-kDa band in samples precipitated with anti-HA serum (indicated with an asterisk) is the same as the one observed when only nsp3 was expressed (Fig. 1C). nsp4 bands and putative nsp4- and nsp6-containing precursor bands were fuzzy, likely due to their relatively large hydrophobic domains. (C and D) HuH-7 cells were transfected with indicated plasmids and analyzed using EM at 24 h posttransfection. Red arrows indicate possible connections between the ER and the cubic membranes. The insets show some areas where double membranes can be observed. M, mitochondria; LD, lipid droplet. Bars, 500 nm.
FIG 5
FIG 5
Cleavage of MERS-CoV nsp3/nsp4 junction is essential for DMV formation. (A) 293T cells were transfected with indicated plasmids and analyzed using Western blotting. nsp3 was detected with anti-SARS-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody. (B to E) HuH-7 cells were transfected with mutant nsp3-4 constructs individually (B and C) or cotransfected with the PLpro domain of nsp3 (D and E) and analyzed using EM. Red arrows point at zippered ER, and in panel E, some putative DMVs are indicated with red asterisks. N, nucleus; M, mitochondria; LD, lipid droplet. Bars, 500 nm.
FIG 6
FIG 6
SARS-CoV nsp3 and nsp4 also suffice to induce DMV formation. (A and B) 293T cells were cotransfected with plasmids encoding SARS-CoV nsp3 and nsp4 and fixed for ET analysis 24 h posttransfection. Two virtual slices (8.5 nm) from reconstructed tomograms (available as Movies S3 and S4, respectively) are shown. Red arrows indicate zippered ER, and red asterisks indicate all the DMVs in this virtual slice that were fully reconstructed in the tomogram. (C and D) 293T cells were cotransfected with plasmids encoding MERS-CoV nsp3 and nsp4 and fixed for conventional EM analysis 24 h posttransfection. DMVs are indicated with red asterisks, and red arrows point at zippered ER. Bars, 250 nm.
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References

    1. Drosten C, Günther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguière AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Müller S, Rickerts V, Stürmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976. doi:10.1056/NEJMoa030747. - DOI - PubMed
    1. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ, SARS Working Group . 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966. doi:10.1056/NEJMoa030781. - DOI - PubMed
    1. van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki AM, Osterhaus AD, Haagmans BL, Gorbalenya AE, Snijder EJ, Fouchier RA. 2012. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio 3:e00473-12. doi:10.1128/mBio.00473-12. - DOI - PMC - PubMed
    1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. doi:10.1056/NEJMoa1211721. - DOI - PubMed
    1. Perlman S, Netland J. 2009. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7:439–450. doi:10.1038/nrmicro2147. - DOI - PMC - PubMed

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