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CORD-19:bd51ebd1d7d5c9161290c1a89d4e3fb7434db6b5 JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
T1 0-91 Sentence denotes A structural analysis of M protein in coronavirus assembly and morphology HHS Public Access
T2 93-101 Sentence denotes Abstract
T3 102-284 Sentence denotes The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles.
T4 285-436 Sentence denotes We investigated how M structure and organization is related to virus shape and size using cryoelectron microscopy, tomography and statistical analysis.
T5 437-561 Sentence denotes We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer.
T6 562-682 Sentence denotes Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature.
T7 683-767 Sentence denotes In contrast, compact M protein is associated with flexibility and low spike density.
T8 768-969 Sentence denotes Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M.
T9 970-1056 Sentence denotes These findings provide insight into how M protein functions to promote virus assembly.
T10 1057-1210 Sentence denotes Cryo-electron microscopy; cryo-electron tomography; pleomorphic virus structure; coronavirus; viral matrix protein M proteins from MHV (Klumperman et al.
T11 1212-1280 Sentence denotes Every type of virus architecture has its own structural constraints.
T12 1281-1435 Sentence denotes In other words, there is a limit to the variation in shape, size or protein configuration that can be realized by a particular set of structural proteins.
T13 1436-1638 Sentence denotes When this tolerance is exceeded, the result becomes uncertain: it is possible that the assembly process may fail, produce misshapen but otherwise infectious particles, or yield non-infectious particles.
T14 1639-1739 Sentence denotes Pleomorphic enveloped viruses represent the most extreme cases of natural variation during assembly.
T15 1740-1913 Sentence denotes Pleomorphic virions can vary considerably in size, as in the case of arenaviruses (Neuman et al., 2005) or shape, as in the case of influenza A virus (Harris et al., 2006) .
T16 1914-2049 Sentence denotes Although this variation has been documented, little is known about how the assembly components shape the overall particle architecture.
T17 2050-2343 Sentence denotes Our understanding of virus assembly has practical implications: a new generation of HIV-1 assembly inhibitors acts at the level of particle architecture by reducing the fidelity of the assembly process (Tang et al., 2003) or by blocking connections between Gag proteins (Sticht et al., 2005) .
T18 2344-2474 Sentence denotes In this study we have chosen to analyze the relationship between composition and architecture for three pleomorphic coronaviruses:
T19 2475-2591 Sentence denotes Mouse hepatitis virus (MHV), Severe acute respiratory syndrome coronavirus (SARS-CoV) and Feline coronavirus (FCoV).
T20 2592-2742 Sentence denotes Recent electron microscopy studies have confirmed that coronavirus particles vary considerably in size, and so can safely be described as pleomorphic.
T21 2743-2968 Sentence denotes However, there is disagreement over the extent of variation in virion shape (Barcena et al., 2009; Beniac et al., 2006; Neuman et al., 2006; Risco et al., 1996) , although a range of morphologies is represented in each study.
T22 2969-3175 Sentence denotes These three coronaviruses make an interesting dataset because each is built from a conserved set of components, but amino acid identity between the homologous structural proteins is typically less than 30%.
T23 3176-3682 Sentence denotes Four structural proteins are important for coronavirus infectivity: the integral membrane protein M adapts a region of membrane for virus assembly and captures other structural proteins at the budding site, the N protein chaperones and protects the viral RNA genome, spikes consisting of three copies of the S glycoprotein promote receptor-binding and membrane fusion, and the small membrane protein E is present in sub-stoichiometric amounts and acts as an enhancer of budding (Hogue and Machamer, 2007) .
T24 3683-3979 Sentence denotes In this study, we will focus on the role of M in assembly and in determining particle morphology. (Machamer and Rose, 1987) , Transmissible gastroenteritis virus, (TGEV) (Klumperman et al., 1994) and Bovine coronavirus (Nguyen and Hogue, 1997) are targeted to the vicinity of the Golgi apparatus.
T25 3980-4518 Sentence denotes Reverse genetic studies and VLP assembly studies suggest that M protein promotes assembly by interacting with viral ribonucleoprotein (RNP) and S glycoproteins at the budding site (de Haan et al., 1999; Escors et al., 2001a; Escors et al., 2001b; Kuo and Masters, 2002; Narayanan et al., 2000; Nguyen and Hogue, 1997; Opstelten et al., 1995; Sturman et al., 1980) , and by forming a network of M-M interactions that is capable of excluding some host membrane proteins from the viral envelope (de Haan et al., 2000; Neuman et al., 2008b) .
T26 4519-4616 Sentence denotes M proteins interact through both the transmembrane domain and endodomain (de Haan et al., 2000) .
T27 4617-4714 Sentence denotes M can also interact with RNA that carries the genomic packaging signal (Narayanan et al., 2003) .
T28 4715-4937 Sentence denotes Coronavirus assembly is then completed at the membrane of a pre-Golgi compartment, as shown most recently in a tomography study of intracellular structures involved in virus replication and assembly (Knoops et al., 2008) .
T29 4938-5061 Sentence denotes Packets of virions are then shuttled out of the cell along the secretory pathway (reviewed in (Hogue and Machamer, 2007) ).
T30 5062-5332 Sentence denotes The minimum requirement for MHV virus-like particle (VLP) production is co-expression of M and E protein (Vennema et al., 1996) , although in some expression systems, the additional co-expression of N increases the efficiency of VLP production (Boscarino et al., 2008) .
T31 5333-5707 Sentence denotes Recent studies have begun to reveal the structure of the coronavirus pre-fusion spike , N protein (Chen et al., 2007; Fan et al., 2005; Huang et al., 2004; Jayaram et al., 2006; Saikatendu et al., 2007; Schutze et al., 2006) , the hemagglutinin-esterase protein, which is found on some group 2 coronaviruses (Zeng et al., 2008) , and the E protein (Pervushin et al., 2009) .
T32 5708-5964 Sentence denotes Also, transmembrane features have been identified as M on SARS-CoV, MHV, FCoV and TGEV particles using cryo-electron microscopy (Neuman et al., 2006) and cryo-electron tomography (Barcena et al., 2009 ), but the structure of M remains poorly characterized.
T33 5965-6179 Sentence denotes The lack of detailed structural and functional information is largely due to its small size, close association with the viral envelope and a tendency to form insoluble aggregates when perturbed (Lee et al., 2005) .
T34 6180-6289 Sentence denotes In this study we have attempted to provide a better understanding of the structure and function of M protein.
T35 6290-6411 Sentence denotes First, we have used cryo-EM and tomography to probe the structure of M in the envelope of MHV, SARS-CoV and FCoV virions.
T36 6412-6500 Sentence denotes Second, we have analyzed the structure of the MHV M protein in VLPs that lack S and RNP.
T37 6501-7062 Sentence denotes This is because identification of M in electron micrographs of virions is complicated by the presence of transmembrane regions of spikes and RNP, and, more importantly, intermolecular interactions could potentially affect coronavirus morphology, including M-M, M-E, M-N, M-S, M-RNA and N-RNA interactions (reviewed in (Hogue and Machamer, 2007) ), palmitin-mediated interactions involving S and E (Boscarino et al., 2008; Lopez et al., 2008; Thorp et al., 2006) , and envelope stretching caused by the packaged helical ribonucleoprotein (Barcena et al., 2009 ).
T38 7063-7249 Sentence denotes Together, these experiments reveal new facets of the structure and function of M, and demonstrate how pleomorphicity can be harnessed to reveal the function of membrane protein networks.
T39 7250-7446 Sentence denotes Growth, purification and imaging of SARS-CoV from Vero-E6 cells, FCoV from AK-D cells and MHV-OBLV60 (Gallagher et al., 1991) ) from DBT cells has been described previously (Neuman et al., 2006) .
T40 7447-7713 Sentence denotes A small plaque mutant derived from MHV-A59, called MHV-sp1, was isolated by plaque purification from the medium from persistently infected 17clone 1 (17cl-1) cells that had survived infection with MHV-A59 and had been passaged more than forty times (Sawicki, 1987) .
T41 7714-7859 Sentence denotes The MHV-sp1 S glycoprotein is not cleaved at the S1-S2 boundary and it is resistant to cleavage by trypsin, unlike the S-glycoprotein of MHV-A59.
T42 7860-7899 Sentence denotes MHV-sp1 was purified by centrifugation.
T43 7900-8057 Sentence denotes First, the virus from 450 ml of infected cell supernatant (~5×10 11 pfu) was pelleted by centrifugation for 3 hours at 24,000 rpm at 4°C using an SW28 rotor.
T44 8058-8162 Sentence denotes The pellet was allowed to dissolve on ice overnight in buffered saline (0.15M NaCl, 20mM HEPES, pH 6.8).
T45 8163-8429 Sentence denotes The 6 ml of the suspended virus was layer on top of linear gradient of 40% (w/w) potassium tartrate, 20mM HEPES, pH 7.4 (bottom) to 15% (w/w) glycerol, 20mM HEPES, pH 7.4 (top) and subjected to isopycnic centrifugation in a SW28 rotor (3 hours at 24,000 rpm at 4°C).
T46 8430-8581 Sentence denotes The resulting milky band of virus was diluted with buffered saline and pelleted by centrifugation for 3 hours at 24,000 rpm at 4°C using an SW28 rotor.
T47 8582-8661 Sentence denotes The pellet was allowed to dissolve on ice overnight in 1 ml of buffered saline.
T48 8662-8804 Sentence denotes VLPs were produced by transfecting HEK-293T cells with pCAGGS expression vectors encoding M, E and N from MHV-A59 (Lokugamage et al., 2008) ).
T49 8805-9061 Sentence denotes Briefly, flasks of nearconfluent cells comprising approximately 1.5 m 2 of total culture area were transfected with plasmid DNA using Lipofectamine and "Plus" reagent (Invitrogen) according to an appropriately scaled version of the manufacturer's protocol.
T50 9062-9319 Sentence denotes Virus-like particles were precipitated from culture medium 48h after transfection using 10% polyethylene glycol and 2.2% w/v NaCl, then further purified by 10%-30% sucrose density gradient ultracentrifugation as described previously (Neuman et al., 2008a) .
T51 9320-9732 Sentence denotes Production and cryo-EM of unilamellar phospholipid vesicles (SUV 100 ) created from a blend of 80 parts 1,2-dioleoyl-sn-glycero-3-phosphocholine, 10 parts 1,2-dioleoyl-snglycero-3-[phosphor-rac-(1-glycerol)], 2 parts 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000], and 1 part 3,3'-dioctadecyloxacarbocyanine perchlorate has been described previously (Kunding et al., 2008) .
T52 9733-9793 Sentence denotes Cryo-EM was done using standard low-dose imaging conditions.
T53 9794-9882 Sentence denotes Images of MHV-OBLV60, VLPs and FCoV were recorded using Leginon (Suloway et al., 2005) .
T54 9883-10032 Sentence denotes Cryo-electron tomography of MHV-sp1 was done using a JEOL JEM-2200FS microscope with energy filter operated at 200 kV and 25,000 times magnification.
T55 10033-10192 Sentence denotes Specimens were tilted along one axis through 140°, from +70° to −70°, and images were recorded to a 4k×4k CCD at a nominal resolution of 5 angstroms per pixel.
T56 10193-10349 Sentence denotes Tomographic reconstruction was performed using the imod software suite (Kremer et al., 1996) by fiducial alignment of 10 nm gold particles in tilted images.
T57 10350-10480 Sentence denotes Two-fold binning was performed at the time of imaging to produce a model with a calibrated resolution of 10.6 angstroms per pixel.
T58 10481-10575 Sentence denotes Details of the equipment and conditions used in conventional cryo-EM are provided in Table 1 .
T59 10576-10678 Sentence denotes Before analysis, image contrast was inverted so that protein density appeared white rather than black.
T60 10679-10811 Sentence denotes We corrected for the effects of phase reversal in the contrast transfer function using the EMAN module ctfit (Ludtke et al., 1999) .
T61 10812-10950 Sentence denotes The images used for two-dimensional reconstructions displayed Thon rings, which indicate the presence of image data, to 8-16 Å resolution.
T62 10951-11095 Sentence denotes Micrographs were filtered in Fourier space to truncate high-frequency image data beyond the last visible Thon ring using the EMAN module proc2d.
T63 11096-11164 Sentence denotes We then selected small images showing M using the EMAN module boxer.
T64 11165-11278 Sentence denotes Image clustering and refinement of class averages was performed using the startnrclasses and classalign2 modules.
T65 11279-11394 Sentence denotes Full contrast transfer function correction was implemented during construction of class averages using classalign2.
T66 11395-11511 Sentence denotes The clearest, most coherent images of M densities were obtained when correction was applied through 17 Å resolution.
T67 11512-11706 Sentence denotes For radial density analysis to assess the location of S, M and the RNP inside the virion, quadrants of particles were selected to minimize the distortion caused by small variations in curvature.
T68 11707-11819 Sentence denotes Quadrants were selected for inclusion in the final average based on clarity and contrast in the envelope region.
T69 11820-12015 Sentence denotes For radial density analysis of M COMPACT and M LONG , radial density maps were constructed by selecting and aligning numerous small wedges centered at the particle edge, which were then averaged.
T70 12016-12251 Sentence denotes Radial density analysis was performed using the SPIDER image analysis suite (Frank et al., 1996) , and was normalized to the brightest and darkest datapoints, which were assigned relative brightness values of 100% and 0%, respectively.
T71 12252-12394 Sentence denotes The signal from the envelope region was used as a fiducial mark for normalization of image intensity and alignment of radial density profiles.
T72 12395-12628 Sentence denotes Two observers recorded two sets each of orthogonal measurements of the longest and shortest visible diameter of each virion or VLP, so that contributions of observer bias and measurement variation could be factored into our analysis.
T73 12629-12840 Sentence denotes Average diameter (d AVG ) was taken as a measure of particle size, and a ratio of the longest to the shortest visible diameter (d MAX /d MIN ), was taken as a measure of particle shape ( Supplementary Fig. S1 ).
T74 12841-12919 Sentence denotes Diameters were measured to the outer edge of the membrane, leaving out spikes.
T75 12920-13085 Sentence denotes Particles were excluded if part of the membrane was outside the captured image area, or if the membrane overlapped with the carbon support layer or another particle.
T76 13086-13251 Sentence denotes Multilamellar, overlapping or tubular SUV 100 particles, and exosomal vesicles that appeared to have inner contents or membrane-embedded proteins were also excluded.
T77 13252-13393 Sentence denotes To assess the precision of our measurements, the distance between lipid bilayer headgroup densities was measured repeatedly by two observers.
T78 13394-13542 Sentence denotes The distance between the brightest parts of the lipid bilayer on vesicles matched expected results to within 1 nm (Nagle and Tristram-Nagle, 2000) .
T79 13543-13763 Sentence denotes Measurements of the longest and shortest diameter of vesicles were less precise, perhaps due to combined errors in measurement and identification of the maximum and minimum particle diameter (see Supplementary Fig. S2 ).
T80 13764-13945 Sentence denotes In order to assess whether virus size and shape varied from one preparation to another, we compared cryo-EM images of four preparations of MHV-OBLV60 and one preparation of MHV-sp1.
T81 13946-14144 Sentence denotes MHV-OBLV60 virions were fixed with 1% phosphate-buffered formalin (pH 7.0) before imaging; MHV-sp1 virions were not fixed because of differences in local biosafety regulations between EM facilities.
T82 14145-14232 Sentence denotes Particle diameter was similar for all five MHV preparations (Supplementary Figure S3) .
T83 14233-14391 Sentence denotes The shape of particles in two OBLV60 preparations was significantly more elongated than the other three, so these particles were excluded from shape analysis.
T84 14392-14614 Sentence denotes Before marking particles as spike-depleted or containing an envelope thickness anomaly, we revised our criteria empirically by repeatedly examining images of SARS-CoV particles and tracking the agreement between observers.
T85 14615-14706 Sentence denotes The criteria were revised until inter-observer agreement was consistently greater than 80%.
T86 14707-15050 Sentence denotes The final criteria for categorization were: "Does closely-packed spike decoration extend around at least half of the particle edge?" and, "Is an abnormally thin region of no less than one-twelfth the virion circumference visible in the M density layer?" Statistics describing inter-observer agreement are presented in Supplementary Figure S2 .
T87 15051-15231 Sentence denotes Complementary DNA was generated by reverse transcriptase PCR, and a fragment encoding amino acids 107-221 of the SARS-CoV-Tor2 M-protein was cloned into pET46Ek/LIC (Novagen, USA).
T88 15232-15342 Sentence denotes The vector encodes an N-terminal poly-histidine tag and a flanking enterokinase cleavage site for tag removal.
T89 15343-15506 Sentence denotes Expression of M 107-221 was achieved in BL21 (DE3) E. coli (Invitrogen, USA) and induced at an OD600 of 0.6-0.8 with 1mM IPTG followed by overnight growth at 18°C.
T90 15507-15620 Sentence denotes The majority of the expressed protein was present as insoluble inclusion bodies which were subsequently refolded.
T91 15621-15820 Sentence denotes Bacteria were lysed at 4°C using an EmulsiFlex® C-3 cell disruptor (Avestin, Canada) at 15 kpsi in lysis buffer (20mM Tris-HCl, 100mM sodium chloride, 10mM dithiothreitol, 1% Triton X-100 at pH 7.0).
T92 15821-15917 Sentence denotes The insoluble fraction containing the inclusion bodies was isolated by low-speed centrifugation.
T93 15918-16087 Sentence denotes Pellets containing the inclusion bodies were resuspended in lysis buffer and then pelleted by centrifugation three times to remove any soluble material that was present.
T94 16088-16274 Sentence denotes The pellets of insoluble material were then washed 3 times, as above, with wash buffer (20mM Tris-HCl, 100mM sodium chloride, 10mM dithiothreitol at pH 7.0) to remove residual detergent.
T95 16275-16512 Sentence denotes The resultant white pellet was dissolved in guanidine buffer (6M guanidine hydrochloride, 50mM Tris-HCl, 10mM dithiothreitol, 1mM EDTA at pH 8.0) to a final concentration of 15 mg/ml as determined by Bradford protein assay (BioRad, USA).
T96 16513-16598 Sentence denotes Solubilized M 107-221 inclusion bodies were refolded using the rapid dilution method.
T97 16599-16822 Sentence denotes Solubilized inclusion body protein was rapidly added dropwise to refolding buffer (50mM HEPES, 200mM sodium chloride, 1M NDSB-201, 10mM beta-mercaptoethanol at pH 7.0, 4°C) to give a final protein concentration of 0.1mg/ml.
T98 16823-16942 Sentence denotes The protein was allowed to refold for 1-3 h before being applied to a 5 ml nickel affinity column (GE Healthcare, USA).
T99 16943-17178 Sentence denotes The column was washed with 5 column volumes of binding buffer (50mM HEPES, 200mM sodium chloride, 10 mM beta-mercaptoethanol at pH 7.0) and refolded protein was eluted in binding buffer supplemented with 250 mM imidazole and 1 mM EDTA.
T100 17179-17390 Sentence denotes Further purification was achieved by passing 2 ml samples of the eluted protein over a pre-equilibrated Superdex™ 75 16/60 size exclusion column (20mM HEPES, 200mM sodium chloride, 5mM dithiothreitol at pH 7.0).
T101 17391-17518 Sentence denotes The purified monomeric protein was concentrated to 1.8 mg/ml using 0.5 ml Ultrafree Biomax 5kDa concentrators (Millipore, USA).
T102 17519-17569 Sentence denotes The protein was >95% pure as assessed by SDS-PAGE.
T103 17570-17665 Sentence denotes Purified protein was stored in 20mM HEPES, 200mM sodium chloride, 5mM dithiothreitol at pH 7.0.
T104 17666-17828 Sentence denotes The stoichiometry of expressed M endodomain was assessed using perfluoro-octanoic acid polyacrylamide gel electrophoresis (PFO-PAGE; (Ramjeesingh et al., 1999) ).
T105 17829-17987 Sentence denotes To do this, 20 µl, 10 µl, 5 µl or 2 µl of purified protein was made up to 20 µl total by adding 20mM HEPES, 200mM sodium chloride, 5mM dithiothreitol, pH 7.0.
T106 17988-18160 Sentence denotes Samples were incubated at either 4°C or 37°C for 1 h then separated by electrophoresis on precast 4-20% acrylamide gels in Tris-glycine buffer containing 0.5% (wt/vol) PFO.
T107 18161-18217 Sentence denotes Protein was detected by SYPROruby staining (Invitrogen).
T108 18218-18426 Sentence denotes The main problem with using cryo-EM to investigate protein structure is limited resolution, which usually does not allow for the structure to be interpreted by an atomic model (Stewart and Grigorieff, 2004) .
T109 18427-18520 Sentence denotes As a consequence, it can be difficult to identify small features such as M in cryo-EM images.
T110 18521-18777 Sentence denotes Previous studies have used what is known about protein size, topology and function to infer the structure of spikes and RNP (Barcena et al., 2009; Beniac et al., 2006; Cavanagh, 1983; Davies and Macnaughton, 1979; Neuman et al., 2006; Risco et al., 1996) .
T111 18778-18933 Sentence denotes However, none of these studies produced a clear view of M, which is obscured in micrographs by the viral membrane, the C-termini of S proteins and the RNP.
T112 18934-19175 Sentence denotes To better understand the structure and organization of M, we examined by cryo-EM ( Fig. 1a -c) and cryo-electron tomography (Figure 1d ), coronavirus particles, vesicles, VLPs containing only M and E, and VLPs containing M, E and N proteins.
T113 19176-19379 Sentence denotes In purified virus preparations we also found a small number of apparently protein-free empty vesicles, which presumably were released from infected cells, and which we refer to in this study as exosomes.
T114 19380-19554 Sentence denotes Viruses and VLPs had thicker envelope regions than vesicles or exosomes due to the presence of M protein, as previously reported (Barcena et al., 2009; Neuman et al., 2006) .
T115 19555-19648 Sentence denotes The purification process used for MHV-sp1 also produced some free, spike-decorated envelopes.
T116 19649-19815 Sentence denotes Free envelopes appeared thicker than ordinary phospholipid bilayers, suggesting that M was still present, but did not contain any trace of the RNP core ( Figure 1d ).
T117 19816-20015 Sentence denotes To determine the boundaries of packaged M (Fig. 2a) , we examined radial density maps from groups of 18-22 empty exosomes, EM and EMN VLPs, low-spike and normal virus particles, as shown in Fig. 2b .
T118 20016-20164 Sentence denotes To identify individual components, we subtracted one set of appropriately scaled and aligned radial density data from another, as shown in Fig. 2c .
T119 20165-20435 Sentence denotes From these data we determined that the endodomain of M extended inward ~8 nm from the highest-density point of the outer membrane leaflet, in agreement with published measurements of MHV M protein from cryo-EM and tomography (Barcena et al., 2009; Neuman et al., 2006) .
T120 20436-20588 Sentence denotes The ectodomain of M did not give rise to a detectable radial density signal, suggesting that it may either be disordered or tightly membrane-associated.
T121 20589-20782 Sentence denotes An area of consistent, high density which extended from 10 nm to 30 nm inside the particle was attributed to RNP, consistent with recent measurements by Barcena et al., (Barcena et al., 2009 ).
T122 20783-20948 Sentence denotes Fig. 2d shows that the variability in radial density was low from the virus exterior through the RNP feature, but innermost parts of the virion were highly variable.
T123 20949-21034 Sentence denotes Taken together, these data show the extent of M and M-linked RNP inside the particle.
T124 21035-21113 Sentence denotes Viral particles were examined closely to identify individual M protein shapes.
T125 21114-21240 Sentence denotes On most particles, M at the virion edge resembled tightly packed lines crossing the membrane and contacting the RNP (Fig. 3) .
T126 21241-21363 Sentence denotes Small M-free areas which may represent budding scars were occasionally visible at the edge of virions and VLPs (Fig. 3a) .
T127 21364-21525 Sentence denotes The observation that M remained tightly packed even on particles with excess M-free membrane provides structural validation of the existence of M-M interactions.
T128 21526-21663 Sentence denotes The observation that M densities appear to make contact with the RNP core likewise can be taken as evidence of M-N or M-RNA interactions.
T129 21664-21793 Sentence denotes On a few particles, some of the M-like protein had a compact, blurred appearance and did not appear to make contact with the RNP.
T130 21794-21871 Sentence denotes We have called the common form M LONG and the short, blurred form M COMPACT .
T131 21872-22120 Sentence denotes Viral spikes can be seen on both M LONG and M COMPACT , but not on M-free membranes ( In order to express M conformation numerically, we performed radial density analysis of small sections at the particle edge where M LONG or M COMPACT was visible.
T132 22121-22350 Sentence denotes The M endodomain density consisted of the tail region, extending 6 to 8 nm into the particle where the differences between M conformations were most apparent, and the denser body region, extending 3.5 to 5.5 nm into the particle.
T133 22351-22569 Sentence denotes After subtracting the density of the ice outside the particle to correct for differences in background and scaling image brightness to a common mean, the ratio of M tail to middle was calculated for each virion or VLP.
T134 22570-22734 Sentence denotes We found that the density in the "tail" region of M COMPACT was similar to the density of the background ice, and the peak M endodomain was higher than for M LONG .
T135 22735-22906 Sentence denotes To express M conformation more simply, we subtracted the background from the tail and body densities and calculated tail to body ratios for M LONG and M COMPACT (Fig 3i) .
T136 22907-23077 Sentence denotes We found that the difference in tail to body ratios was statistically significant (T test, P = 0.014 for FCoV, P = 2.0 × 10 −17 for SARS-CoV and P = 3.0 × 10 −6 for MHV).
T137 23078-23285 Sentence denotes This suggests that the two forms of M represent different conformations of the same peptide chain, and the distribution of density at the virion edge can be used as an approximate readout for M conformation.
T138 23286-23437 Sentence denotes We next examined M proteins in more detail by classifying, aligning and averaging similarlooking regions from particle edges to produce class averages.
T139 23438-23622 Sentence denotes M LONG resembles a dagger, with the ectodomain "pommel" resting on the outer membrane leaflet and the tip of the endodomain "blade" extending ~8 nm and contacting the RNP (Fig. 4a-b) .
T140 23623-23780 Sentence denotes The endodomains of adjacent M LONG particles appeared to contact each other, suggesting that M LONG -M LONG interactions are mediated by the endodomain of M.
T141 23781-23949 Sentence denotes M LONG on EM VLPs appeared similar to M on virions, thus confirming that M LONG is formed by M in the absence of the other high-copy virion proteins S and N (Fig. 4b) .
T142 23950-24033 Sentence denotes Some class averages of M LONG also showed a distinct tilt relative to the membrane.
T143 24034-24253 Sentence denotes The endodomain of M LONG was resolved into two globular components with crisp borders between adjacent molecules, while the endodomain of M COMPACT appeared as an indistinct ellipsoid and extended only ~6 nm (Fig. 4a) .
T144 24254-24493 Sentence denotes The two globular components of the endodomain were not distinctly observed in class averages from EM VLPs (Fig 4b) , suggesting that either the conformation is only formed in the presence of N or that N forms part of the two-lobed M LONG .
T145 24494-24594 Sentence denotes M LONG and M COMPACT were also visible in virtual slices through tomographic reconstructions of MHV.
T146 24595-24837 Sentence denotes The difference in apparent length was most evident on tomographic slices through free envelopes, which showed thick convex M LONG membranes near the center giving way to thinner variably curved M COMPACT membranes near the edges (Fig. 5a-b) .
T147 24838-24941 Sentence denotes M LONG was spaced 4 to 5 nm apart in unprocessed cryo-EM images and edge view class averages (Fig. 6) .
T148 24942-25151 Sentence denotes In axial views of M taken from the centers of EM and EMN VLPs, M sometimes appeared to form small ordered regions (Fig. 6a) which could be classified, aligned and averaged to produce class averages (Fig. 6b) .
T149 25152-25240 Sentence denotes M spacing in axial views was generally consistent with spacing in edge views (Fig. 6c) .
T150 25241-25560 Sentence denotes While axial class averages suggest that M packing generally approximates a rhombus with sides of 4.0 and 4.5 nm and an interior angle of about 75 degrees, the amount of heterogeneity in the unprocessed images is more consistent with a loosely-ordered network of M, as opposed to a rigid two-dimensional protein lattice.
T151 25561-25788 Sentence denotes Edge view class averages of M LONG from MHV, FCoV and SARS-CoV virions appeared about twice as large as expected for a single copy of the M protein, based on the partial specific volume of folded protein (Harpaz et al., 1994) .
T152 25789-25973 Sentence denotes This can be seen by comparing class averages showing M (Fig. 4b) with projections of ellipsoids with the same spacing as M, and the volume of one, two or three copies of M, (Fig. 4c ).
T153 25974-26080 Sentence denotes This suggested that each M LONG and M COMPACT is large enough to contain at least two copies of M protein.
T154 26081-26168 Sentence denotes We next attempted to investigate the stoichiometry of purified M protein biochemically.
T155 26169-26289 Sentence denotes Attempts to express SARS-CoV M protein in E. coli and baculovirus expression systems produced only insoluble aggregates.
T156 26290-26520 Sentence denotes The largest M construct which we were able to express and purify consisted of residues 107 to 221 from SARS-CoV M, which covers the area of the endodomain from just after the third predicted transmembrane region to the C-terminus.
T157 26521-26606 Sentence denotes M 107-221 was expressed as an aggregate initially, but became soluble upon refolding.
T158 26607-26948 Sentence denotes Since a fusion protein incorporating the endodomain of MHV M was shown to coimmunoprecipitate with full-length M protein (de Haan et al., 2000) , and class averages suggested that adjacent M LONG dimers make contact via two parts of the endodomain, we decided to investigate the oligomerization of M 107-221 as a surrogate for full-length M.
T159 26949-27160 Sentence denotes Soluble endodomain incubated in saline at 37°C was present as monomers and aggregates as well as units of two, four and six, while endodomain incubated at 4°C remained mostly monomeric (Supplementary Figure 4) .
T160 27161-27239 Sentence denotes These observations support the interpretation that M functions as a homodimer.
T161 27240-27357 Sentence denotes In solution, the shape of a vesicle is determined by the interplay of opposing forces (Zhongcan and Helfrich, 1987) .
T162 27358-27413 Sentence denotes The ground state for vesicles in solution is spherical.
T163 27414-27530 Sentence denotes Forces imparted by fluid motion or sample freezing in preparation for cryo-EM can temporarily distort vesicle shape.
T164 27531-27751 Sentence denotes However, as a particle becomes less spherical the effects of hoop stress will increase, leading either to structural failure of the vesicle wall or driving the vesicle back to a more toward spherical, lower-stress shape.
T165 27752-27965 Sentence denotes Since enveloped virus particles and are essentially protein-decorated vesicles, we reasoned that their shape should be similar to the shape of vesicles unless modified by the effects of viral protein interactions.
T166 27966-28166 Sentence denotes To test whether viral proteins significantly affected the shape of viral particles, we compared the shape of virions, VLPs and small unilamellar vesicles of the same size (SUV 100 ) in cryo-EM images.
T167 28167-28338 Sentence denotes In our images, vesicles ranged from round to ellipsoidal, with the average particle having a long axis 1.08 times the length of the shortest axis ( Supplementary Fig. 1 ).
T168 28339-28424 Sentence denotes The shape of MHV and EM VLPs did not differ significantly from the shape of vesicles.
T169 28425-28550 Sentence denotes However, EMN VLPs were significantly rounder than the combined population of SUV 100 and endosomal vesicles (ANOVA, p=0.005).
T170 28551-28731 Sentence denotes Although EM particles were rounder on average than EMN particles or vesicles, the difference was not significant due to the small sample size and heterogeneity of the EM particles.
T171 28732-28892 Sentence denotes This indicates that the protein component of EMN VLPs provides significant resistance to the deformative forces that affected vesicles under cryo-EM conditions.
T172 28893-28990 Sentence denotes We next sought an explanation for how E, M and N proteins can make spherical vesicles more rigid.
T173 28991-29233 Sentence denotes N proteins can dimerize (Chen et al., 2007) and pack helically in the presence (Barcena et al., 2009 ) and absence (Saikatendu et al., 2007) of RNA, but neither of these interactions alone would be expected to produce a spherical arrangement.
T174 29234-29345 Sentence denotes E protein is small and not abundant enough in viral particles to be a convincing explanation for overall shape.
T175 29346-29460 Sentence denotes In contrast, M is abundant, interacts with other M proteins and with membranes, which are approximately spherical.
T176 29461-29541 Sentence denotes We therefore hypothesized that particle shape is controlled primarily through M.
T177 29542-29660 Sentence denotes A striking difference between M LONG and M COMPACT is that M LONG appears to contact the RNP while M COMPACT does not.
T178 29661-29822 Sentence denotes In cryo-EM images, the internal RNP appeared to be pulled away from the particle edge where either M COMPACT or M-free membrane is present, as seen in Figure 3 .
T179 29823-29925 Sentence denotes Thus, M COMPACT and M-free membranes can both be viewed as local disruptions of the M-RNP interaction.
T180 29926-30219 Sentence denotes To test the hypothesis that M-mediated interactions control particle shape, we compared the shape of particles in which M LONG appeared to form a complete ring at the particle edge to the shape of particles in which the ring of M LONG was interrupted by either a M-free membrane or M COMPACT .
T181 30220-30396 Sentence denotes Interruption of M LONG at the particle edge was associated with significant particle elongation in SARS-CoV (ANOVA, P = 5×10 −12 ), FCoV (P = 0.01) and EM (P = 0.03) particles.
T182 30397-30465 Sentence denotes A similar but non-significant trend was observed for MHV (Fig. 7a) .
T183 30466-30562 Sentence denotes While a relative increase in M anomalies was correlated with particle elongation, we noted that:
T184 30563-30742 Sentence denotes The relative abundance of particles with interrupted M was quite different in the three coronaviruses, while the frequency of M anomalies was similar in MHV, EM and EMN particles.
T185 30743-30987 Sentence denotes This result shows that an uninterrupted layer of M LONG is a marker for spherical morphology, but suggests that there may be inherent differences in the interaction affinities and membrane-bending properties of different coronavirus M proteins.
T186 30988-31158 Sentence denotes If only M LONG is associated with coronavirus-like membrane curvature, it is implicit that the ratio of M LONG to M COMPACT should be related to local membrane curvature.
T187 31159-31323 Sentence denotes To test this, we selected four parts of each particle edge, which were centered on either side of the longest and shortest particle diameter as shown in Figure 7b .
T188 31324-31429 Sentence denotes Two independent observers counted the number of M dimers present and marked them as M LONG or M COMPACT .
T189 31430-31627 Sentence denotes The ratio of M LONG to M COMPACT was about the same at all of the virion ends, but the proportion of M marked as M LONG on the flatter sides decreased as particles became more elongated (Fig. 7c) .
T190 31628-31704 Sentence denotes From these results we conclude that clusters of M LONG mark round membranes.
T191 31705-31894 Sentence denotes We noted that some samples of purified MHV appeared quite ellipsoidal while others were mostly round, while virion size was similar in all of the MHV preparations ( Supplementary Fig. 3 ).
T192 31895-32007 Sentence denotes Purification over sucrose density gradients would at least temporarily expose viral particles to osmotic stress.
T193 32008-32097 Sentence denotes Likewise, centrifugation would necessarily expose virus particles some mechanical stress.
T194 32098-32181 Sentence denotes We suspected that in some cases the purification process might change virion shape.
T195 32182-32358 Sentence denotes To investigate whether virion shape could be changed experimentally after purification, one sample of purified virus was resuspended in HEPES-buffered 0.9% saline buffer, pH 7.
T196 32359-32585 Sentence denotes Half the sample was kept in the resuspension buffer, while the other half was acidified to pH 5 for 5 minutes in order to simulate pH changes that might occur during entry via the endosomal route, and then re-buffered to pH 7.
T197 32586-32707 Sentence denotes The shape and size of native MHV (Prep 1 in Supplementary Fig. 3 ) and acid-pulsed MHV (Prep 3) were examined by cryo-EM.
T198 32708-32807 Sentence denotes Overall particle shape did not change significantly as a result of acidification (ANOVA, P = 0.93).
T199 32808-32996 Sentence denotes However, the relative proportion of particles which appeared to have at least one flattened edge increased significantly after acid-treatment (Fisher's exact test, P = 0.0001; Fig. 8a-c) .
T200 32997-33217 Sentence denotes In some flat-edged particles, the interior RNP appeared to have a crisp edge, which was more distant from the membrane than RNP in native particles and M had the blurred appearance characteristic of M COMPACT (Fig. 8b) .
T201 33218-33443 Sentence denotes Radial density plots and M tail to body ratios demonstrate that the altered M which was more frequently found at flat edges of acidtreated particles strongly resembled M COMPACT from native MHV particles (Fig. 8d- (Fig. 8e ).
T202 33444-33544 Sentence denotes This suggests that intracellular N plays an important role in the formation or packaging of M LONG .
T203 33545-33644 Sentence denotes Previous studies have shown that spikes are dispensable for assembly but essential for infectivity.
T204 33645-33964 Sentence denotes It was therefore surprising to find that spikeless EM and EMN VLPs were significantly larger than virions ( Supplementary Fig. 1 ) and rare viral particles on which no spikes were visible were significantly larger than spike-decorated virions (Fig. 9a) , suggesting that spike incorporation was linked to envelope size.
T205 33965-34143 Sentence denotes Since the size of the virus envelope is fixed at the scission stage of the budding process, we inferred that spike incorporation is linked to factors which produce small virions.
T206 34144-34331 Sentence denotes To determine which factors were linked to particle size, we measured MHV VLPs in cryo-EM images and MHV virions in tomograms, where spike decoration could be assessed in three dimensions.
T207 34332-34474 Sentence denotes MHV EM VLPs (93 nm average diameter) were larger than EMN VLPs (91 nm) and spikeless MHV in tomograms (91 nm) and spike-decorated MHV (88 nm).
T208 34475-34712 Sentence denotes Viral particle size decreased as particles approached the full complement of proteins and RNP ( Supplementary Fig. 1) , suggesting that small virions are produced as a result of the interplay between all viral components during assembly.
T209 34713-34816 Sentence denotes A few SARS-CoV particles appeared to have clusters of spikes at one or two spots on the viral envelope.
T210 34817-34974 Sentence denotes Spike clusters were significantly associated with the curved ends of ellipsoidal particles (Fig. 9b) , which we had previously found to be marked by M LONG .
T211 34975-35042 Sentence denotes We therefore concluded that M LONG is a marker of spike decoration.
T212 35043-35205 Sentence denotes Here we demonstrate that a network of M has intrinsic membrane-bending properties, as recently demonstrated for the HIV-1 Gag polyprotein (Carlson et al., 2008) .
T213 35206-35360 Sentence denotes We report that M is functionally dimeric in viral particles, and the membrane-altering properties of M depend on interactions with other viral components.
T214 35361-35682 Sentence denotes Our analysis suggests that two types of M-M interactions should be considered in future structural studies: interactions which maintain the M dimer and may occur throughout the protein, and interactions between dimers which are probably mediated by the endodomains, which form a matrix-like layer underneath the membrane.
T215 35683-35872 Sentence denotes In several ways, the function of coronavirus M appears be analogous to that of influenza A virus M1 protein, which shows pH-specific differences in membrane-bending (Ruigrok et al., 1992) .
T216 35873-36054 Sentence denotes Crystallography studies have also revealed that M1 proteins have similar structures at acidic and neutral pH, but differ in the way protein monomers interact (Harris et al., 2001) .
T217 36055-36265 Sentence denotes M of all coronaviruses appears to adopt an N-ecto/C-endo topology (Armstrong et al., 1984) , but transmissible gastroenteritis coronavirus M also adopts an alternate N-endo/Cecto topology (Risco et al., 1995) .
T218 36266-36372 Sentence denotes We initially considered that the difference between M LONG and M COMPACT might be due to altered topology.
T219 36373-36547 Sentence denotes The N-ecto/C-ecto topology proposed for TGEV M involves part of the endodomain forming a fourth transmembrane span, with part of the C-terminus exposed on the virion surface.
T220 36548-36693 Sentence denotes Since the pretransmembrane region of M is much smaller than the post-transmembrane region, an upsidedown topology should be visually distinctive.
T221 36694-36782 Sentence denotes However, the endodomains of M LONG and M COMPACT were of similar size in class averages.
T222 36783-37075 Sentence denotes Instead, we prefer an interpretation in which M COMPACT and M LONG are conformationally distinct homodimers of N-ecto/C-endo topology, and the difference in appearance is due to a conformational change that either stretches (M LONG ) or collapses (M COMPACT ) the structure of the endodomain.
T223 37076-37371 Sentence denotes Prior to this study, the existence of reduction-sensitive complexes with a molecular weight consistent with approximately 2, 4 and 8 copies of M had been reported for HCoV-229E (Arpin and Talbot, 1990) , but formation of a discrete M oligomer had not been demonstrated for any other coronavirus.
T224 37372-37632 Sentence denotes The coronavirus M dimer would appear to be functionally equivalent to the heterodimeric complex of the triple-spanning membrane proteins GP5 and M which is essential for assembly of Equine arteritis virus (Snijder et al., 2003) , a distantly related nidovirus.
T225 37633-38034 Sentence denotes Further study of nidovirus ultrastructure is needed, but these observations coupled with the presence of one or more predicted three-transmembrane protein genes in every known coronavirus (M, but also SARS 3A and FCoV 3C), arterivirus (M and GP5), bafinivirus (GP4/M) and torovirus (M) genome suggests that dimeric complexes of triplespanning membrane proteins may be a hallmark of nidovirus assembly.
T226 38035-38155 Sentence denotes While examining virions, we noticed that particles were most often either entirely spikedecorated or entirely spikeless.
T227 38156-38385 Sentence denotes Further examination revealed that spikeless particles were significantly larger than spike-decorated particles, demonstrating that spikelessness resulted from a defect in assembly, not by accidental shearing or fusion activation.
T228 38386-38470 Sentence denotes This led to a closer examination of SARS-CoV particles with patchy spike decoration.
T229 38471-38619 Sentence denotes Spike patches were most often found at the ends of elongated particles, where M LONG is common, suggesting that M LONG mediates spike incorporation.
T230 38620-38750 Sentence denotes Figure 10a shows a conceptual model of an elongated SARS-CoV particle with spikes decorating both ends, where M LONG is plentiful.
T231 38751-39002 Sentence denotes The association of small clusters of S with M LONG was unexpected, but we were unable to determine whether the missing spikes were not incorporated, or were present in an elongated disordered conformation, as would be expected after fusion activation.
T232 39003-39098 Sentence denotes Another possible explanation for this phenomenon is that incorporated spikes stabilize M LONG .
T233 39099-39259 Sentence denotes There is some evidence to link the endodomain, which appears to undergo the most noticeable change between M LONG and M COMPACT , to incorporation of S protein.
T234 39260-39429 Sentence denotes While the transmembrane region of M is important for M-S interactions, one residue of the M endodomain has been implicated in incorporation of S (de Haan et al., 1999) .
T235 39430-39601 Sentence denotes Mutation of a conserved tyrosine residue at position 211, near the N interaction site, does not affect VLP production but prevents S incorporation (de Haan et al., 1999) .
T236 39602-39713 Sentence denotes The C-terminus of the M endodomain shows the most profound structural difference between M LONG and M COMPACT .
T237 39714-39821 Sentence denotes This suggests that tyrosine-211 may be important for M LONG stability or for conversion between conformers.
T238 39822-39928 Sentence denotes Further study is needed to determine the structural basis for coronavirus M, N and S protein interactions.
T239 39929-40076 Sentence denotes By comparing M proteins on virus particles and VLPs, we identified N, S and genomic RNA as factors that increase the ratio of M LONG to M COMPACT .
T240 40077-40310 Sentence denotes Properties attributed specifically to M LONG , which include membrane rigidity, uniform curvature and spike incorporation, appear to be geared to assembly of infectious virus, and appear to be opposed by the properties of M COMPACT .
T241 40311-40480 Sentence denotes We would therefore hypothesize that factors or treatments which increase the relative abundance of M LONG predict structural success, marked by increased virion fitness.
T242 40481-40582 Sentence denotes Further work is needed to address the relationship between internal virion structure and infectivity.
T243 40583-40856 Sentence denotes Protein spacing data presented here and previously (Neuman et al., 2006) suggests that 8 dimeric M densities can accommodate a maximum of four N proteins and one trimeric spike protein, thus forming an 8M 2 :4N:1S 3 unit at the virion surface (see the boxed region in Figs.
T244 40857-40864 Sentence denotes 10b-c).
T245 40865-41104 Sentence denotes We previously reported that the minimum spacing between spikes on highly decorated SARS-CoV particles was ~14-15 nm, which is about 4-5 nm farther apart than would be expected based on the ~10 nm width of each spike (Neuman et al., 2006) .
T246 41105-41326 Sentence denotes Fourier transformation revealed a ~13-18 nm −1 frequency signal in tomographic projections of spike-decorated MHV, but not spikeless MHV, confirming that spike spacing for MHV-sp1 and SARS CoV is similar (data not shown).
T247 41327-41469 Sentence denotes The diagrams in Figures 10b and 10c show a packing model in which adjacent spikes are incorporated at anchor points across the M LONG network.
T248 41470-41682 Sentence denotes Each M dimer could potentially interact with a spike, but the closest packing that could be achieved in this model would be ~14-15 nm apart, based on the bulk of each spike and the nearest available anchor point.
T249 41683-41804 Sentence denotes To test the quality of this model, we counted the number of spikes on three-dimensional reconstructions of MHV particles.
T250 41805-41922 Sentence denotes These particles showed an average of 74 spikes per particle, which gives an approximate inter-spike spacing of 17 nm.
T251 41923-41966 Sentence denotes Our model predicts ~90 spikes per particle.
T252 41967-42089 Sentence denotes Low spike incorporation in areas of M COMPACT may explain some of the difference between predicted and actual spike count.
T253 42090-42191 Sentence denotes The protein spacing model in Fig. 10b -c shows an average spacing of 4-5 nm between M protein dimers.
T254 42192-42324 Sentence denotes Using the M spacing data for each virus (Fig. 6c) , this would give ~1100 M 2 molecules per average SARS-CoV, MHV and FCoV particle.
T255 42325-42452 Sentence denotes We are unable to directly count the number of M features per particle because of the resolution of the reconstructed tomograms.
T256 42453-42617 Sentence denotes However, the ratio of M to N in purified coronavirus particles has been well studied, and provides an indirect way to test the validity of our proposed M 2 spacing.
T257 42618-42840 Sentence denotes Estimated ratios of M to N protein in purified coronaviruses range from about 3M:1N (Cavanagh, 1983; Escors et al., 2001b) to 1M:1N (Hogue and Brian, 1986; Liu and Inglis, 1991) , giving 730 to 2200 N molecules per virion.
T258 42841-42909 Sentence denotes That works out to one N protein per 14-40 nucleotides of the genome.
T259 42910-43163 Sentence denotes By comparison, the nucleoproteins of rabies virus and vesicular stomatitis virus are of similar molecular weight to coronavirus N proteins, and have been demonstrated to bind 9 nucleotides per nucleoprotein (Albertini et al., 2006; Green et al., 2006) .
T260 43164-43304 Sentence denotes If, as suggested by Barcena et al., (Barcena et al., 2009) , M dimers are spaced 6.5 nm apart, results of 60-200 nt/N molecule are obtained.
T261 43305-43457 Sentence denotes In the model of coronavirus assembly shown in Fig. 10d , M is shown as a mixed population of M LONG and M COMPACT in the endoplasmic reticulum membrane.
T262 43458-43552 Sentence denotes M is then transported and interacts with other viral membrane proteins at the site of budding.
T263 43553-43652 Sentence denotes The arrival of the ribonucleoprotein acts as a catalyst for the conversion of M COMPACT to M LONG .
T264 43653-43822 Sentence denotes The M LONG densities bend the membrane to form a sphere around the ribonucleoprotein, with the size of the sphere inversely related to the relative abundance of M LONG .
T265 43823-43908 Sentence denotes Supplementary Figure 5 relates virus particle size to membrane curvature per M dimer.
T266 43909-44057 Sentence denotes We hypothesize that factors that interact with M such as N, S and genomic RNA could decrease particle size by limiting variation membrane curvature.
T267 44058-44235 Sentence denotes After budding and release, environmental stress could convert some regions of M LONG back to M COMPACT , allowing the RNP to form longer helices and causing particle elongation.
T268 44236-44295 Sentence denotes The function of E in virogenesis remains poorly understood.
T269 44296-44573 Sentence denotes It is interesting that the function and packaging of E and S are dependent on palmitin acylation (Boscarino et al., 2008; Lopez et al., 2008; Thorp et al., 2006) , and further research will be needed to test whether M densities may contain one or more palmitin binding regions.
T270 44574-44709 Sentence denotes Point mutations in E have been shown to result in the assembly of large, elongated, thermolabile MHV particles (Fischer et al., 1998) .
T271 44710-44933 Sentence denotes In light of the model proposed here, we would speculate that these characteristics can be attributed to an overabundance of packaged M COMPACT , suggesting that E plays a role in promoting M LONG formation or incorporation.
T272 44934-45037 Sentence denotes This interpretation could be tested by cryo-EM and tomography of viruses carrying mutations in M and E.
T273 45038-45122 Sentence denotes In this study we described two functionally distinct forms of coronavirus M protein.
T274 45123-45272 Sentence denotes Both types of M visible on viral particles were larger than expected for a single M protein, but consistent with the expected size of two M proteins.
T275 45273-45371 Sentence denotes We demonstrated that M protein endodomains can self-assemble into oligo-dimeric complexes at 37°C.
T276 45372-45512 Sentence denotes We showed that formation of a convex, rigidified viral envelope is dependent on the presence of one form of M, which we have called M LONG .
T277 45513-45654 Sentence denotes Statistical evidence suggests that spike incorporation is linked to particle size, and that spikes cluster in regions where M LONG is common.
T278 45655-45849 Sentence denotes To explain these observations, we proposed a model in which locally-ordered networks of M LONG , stabilized by S, N and possibly E proteins, control particle size and the efficiency of assembly.
T279 45850-46009 Sentence denotes Cryo-electron micrographs of small unilamellar vesicles (A), MHV-like particles (B), three coronaviruses (C) and cryo-electron tomography of MHV (D) are shown.
T280 46010-46103 Sentence denotes The longest and shortest visible diameter of each particle was measured, as shown in panel A.
T281 46104-46192 Sentence denotes Viral particles were distinguished from empty exosomes by the thickness of the envelope.
T282 46193-46258 Sentence denotes A spikeless particle (*) and free viral envelopes (e) are marked.
T283 46259-46469 Sentence denotes Gold particles which were used as fiducial markers in construction of the tomogram are marked with arrows. shown to illustrate the difference in appearance between M LONG and M COMPACT from three coronaviruses.
T284 46470-46636 Sentence denotes Microscopy conditions and particle count. b Conventional cryo-EM images taken at a defocus of −3.3 µm or closer to focus were used for two-dimensional image analysis.
T285 46637-46665 Sentence denotes c Denotes complete tomograms