The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses
Flexible filamentous viruses are ubiquitous plant pathogens that have an enormous impact in agriculture ( Revers and Garcia , 2015 ) . Their infective particles are non-enveloped and flexible rod-shaped virions ( Kendall et al . , 2008 ) and contain several hundreds of copies of a coat protein ( CP ) arranged in a helical fashion protecting a ssRNA of positive polarity , or ( + ) ssRNA ( Kendall et al . , 2008 ) . They are distributed in families Alphaflexiviridae , Betaflexiviridae , Closteroviridae , and Potyviridae , which have different genomic organizations ( particularly different between Potyviridae and the rest of the groups ) and belong to different superfamilies , but it is thought that their CPs have strong evolutionary relationships ( Koonin et al . , 2015 ) . Structural studies of virions by X-ray fiber diffraction and cryoEM have indeed revealed a common architecture for flexible plant viruses . The filaments are 120-130 Å in diameter , and the CPs are arranged following helical symmetry with slightly less than 9 subunits per turn ( Kendall et al . , 2008 ) . This overall arrangement is shared by Soybean mosaic virus ( SMV ) a potyvirus ( from the family Potyviridae ) , and three different potexviruses ( family Alphaflexiviridae ) , Potato virus X ( PVX ) , Papaya mosaic virus ( PapMV ) , and Narcissus mosaic virus ( NMV ) ( Kendall et al . , 2013 , Yang et al . , 2012 , Kendall et al . , 2008 ) . The flexibility of the virions has limited high-resolution structural studies , and most of the previous data were at moderate resolution . Very recently , the cryoEM structure of Bamboo mosaic virus ( BaMV ) , another potexvirus , was determined at 5 . 6 Å ( DiMaio et al . , 2015 ) , and the CP was modeled based on the atomic structure of a truncated version of the CP from Papaya mosaic virus ( PapMV CP ) ( Yang et al . , 2012 ) . The work revealed that N- and C-terminal extensions of the CP mediate viral polymerization and allow for the flexuous nature of the virions . Pepino mosaic virus ( PepMV ) is another potexvirus which has emerged recently ( Jones et al . , 1980 ) , progressing from endemic to epidemic in tomato crops causing severe economic losses worldwide ( Hanssen and Thomma , 2010 ) . PepMV is transmitted by mechanical contact and virions contain a ( + ) ssRNA of about 6 . 4 kb ( Aguilar et al . , 2002 ) . The PepMV CP is strictly required for cell-to-cell movement of the virus ( Sempere et al . , 2011 ) . We present the cryoEM structure of PepMV virions at 3 . 9 Å of resolution . The near-atomic three-dimensional ( 3D ) map allows for accurate modeling of the CP , the viral RNA , and their interaction . In vivo functional studies of several CP mutants confirm the role of several residues in RNA binding and polymerization . Surprisingly , we have also found a clear structural homology between the CP of flexuous viruses and the NP of the genus Phlebovirus , a group of enveloped viruses with a segmented ( - ) ssRNA genome . The NPs from phleboviruses are associated with the viral genome in loose ribonucleoproteins ( RNPs ) ( Raymond et al . , 2012 ) protected inside an envelope , in which inserted glycoproteins construct an icosahedral shell ( Huiskonen et al . , 2009 , Freiberg et al . , 2008 ) . Despite the divergence of both viral groups , CP from potexviruses and NP from phleboviruses have the same all α-helix fold , and their high similarity suggests a horizontal gene transfer event between these evolutionary distant groups of eukaryotic RNA viruses .
We have analyzed by cryoEM PepMV virions isolated from infected Nicotiana benthamiana plants ( Figure 1—figure supplement 1 ) . The 3D map at 3 . 9 Å of resolution ( Figures 1A , B and Figure 1—figure supplement 3 ) was calculated by single particle-based helical image processing implemented in Spring software ( Desfosses et al . , 2014 ) . The cryoEM map reveals a left-handed helix with a diameter of 130 Å and an inner narrow channel of 13 Å , and the structure shows a pitch of 34 . 6 Å and 8 . 7 CP copies per turn . This overall helical arrangement is in agreement with previous works with other potexviruses ( Kendall et al . , 2013 , Yang et al . , 2012 ) , but in the current case and in the recent cryoEM structure of BaMV ( DiMaio et al . , 2015 ) the attained resolutions allowed a clear assignment of the symmetry . Our cryoEM map reaches near-atomic resolution , to date the highest resolution structural data for a flexible filamentous virion , where bulky protein side chains are discernible ( Figure 1C ) . This allowed us to generate an atomic model for PepMV CP ( Figure 1D ) by iterative modeling starting with the atomic structure of PapMV CP ( see 'Materials and methods' ) . The PepMV CP structure thus generated has three major regions: the core; an N-terminal flexible arm; and a C-terminal extension . The first 20 amino acids in the N-terminal side are not included in the atomic model because the region projects outwards , and its density vanishes due to high flexibility . The modeled structure for PepMV CP is , as defined for PapMV CP , an all-helix fold , and the low RMSD between the two structures ( 1 . 5 Å in the core region ) reflects their clear homology ( Figure 1—figure supplement 5 ) . 10 . 7554/eLife . 11795 . 003Figure 1 . CryoEM structure of PepMV and atomic model for its CP . ( A , B ) Renderings of the 3D density map for PepMV that displays a left-handed helical symmetry with 34 . 6 Å of helical pitch ( P ) . The map is seen segmented domain-wise . The cut-away view ( B ) reveals the location of the ssRNA ( red ) . ( C ) Close-up view of a region from the cryoEM map rendered in semi-transparent mode , together with the atomic model calculated for PepMV CP . ( D ) Isolated density for a PepMV CP subunit shown semi-transparent , and representation of the PepMVCP atomic model . Color code for PepMV CP domains: core region , blue; N-terminal arm , purple; and C-terminal extension , yellow . CP , coat protein , PepMV , Pepino mosaic virus , PepMV CP , Pepino mosaic virus coat protein . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 00310 . 7554/eLife . 11795 . 004Figure 1—figure supplement 1 . Electron micrograph of PepMV cryoEM data . Field of an electron micrograph showing several PepMV virions . The black boxes correspond to straight segments of the helices selected for further data processing . PepMV , Pepino mosaic virus . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 00410 . 7554/eLife . 11795 . 005Figure 1—figure supplement 2 . Helical symmetry search . Search for helical symmetry parameters obtained in Spring package ( Desfosses et al . , 2014 ) . Several 3D maps with different helical symmetry parameters are calculated starting with bi-dimensional averaged classes . Re-projections of the calculated 3D maps are then compared with the 2D averages , and their relative cross-correlation coefficients identify possible parameters . We tested a grid with variations in the helical pitch and the number of subunits per turn and calculated low resolution reconstructions using the parameters with higher correlations . The visual inspection of these initial maps lead to the selection of the helical symmetry for PepMV sample ( better definition of CP subunits in the low-resolution map with 8 . 75 copies per turn ) . Further local searches were also performed to refine the symmetry . The high resolution of the final cryoEM map and its structural details confirmed the correct helical parameters . CP , coat protein . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 00510 . 7554/eLife . 11795 . 006Figure 1—figure supplement 3 . Estimation of resolution for the cryoEM map of PepMV virions . Fourier Shell Correlation ( FSC ) calculated between 3D maps from two fully independently processed halves of the data set ( brown line ) and between the atomic model and the cryoEM map ( green line ) . The FSC threshold at 0 . 14 estimates the resolution at 3 . 9 Å . PepMV , Pepino mosaic virus . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 00610 . 7554/eLife . 11795 . 007Figure 1—figure supplement 4 . Local resolution measurement in isolated PepMV CP subunit . Segmented density for a single PepMV CP subunit is rendered in two orientations showing the estimated local resolution . The calculations were performed with the raw cryoEM map , but only one PepMV CP subunit is shown for clarity . The color scale is also rendered . CP , coat protein; PepMV , epino mosaic virus . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 00710 . 7554/eLife . 11795 . 008Figure 1—figure supplement 5 . Comparison between modeled PepMV CP and the atomic structure of PapMV CP . ( A ) Comparison between the built atomic model for PepMV CP ( colored domain wise as in Figure 1 ) and the reported structure for the truncated version of PapMV CP ( colored grey; pdb code 1DOX ( Yang et al . , 2012 ) ) . The ssRNA belongs to the current model for PepMV virion . The RMSD between the structures in the core region is of 1 . 5 Å . ( B ) Comparison of the current model for PepMV CP and the atomic model for BaMV ( colored brown; pdb code 5A2T ( DiMaio et al . , 2015 ) . The RMSD between both structures in the core region is of 3 . 5 Å . The black arrow indicates a discrepancy between both atomic models in their secondary structure . A short helix in 5A2T is a loop in our model and in 1DOX . This region contributes to the RNA-binding surface . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 00810 . 7554/eLife . 11795 . 009Figure 1—figure supplement 6 . Table of some figures of merit for the structure of the modeled PepMV CP . Short table with figures of merit calculated in Molprobity for the validation of the atomic model of PepMV CP . PepMV CP , Pepino mosaic virus coat proteinDOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 009 The high level of structural details of our cryoEM map permits the segmentation of the density for the ssRNA and the analysis of the protein–RNA interactions . The ssRNA runs in a helix of 70 Å in diameter ( Figure 1B ) . Each CP binds five ribonucleotides ( Figure 1D ) , so that the entire genome of PepMV would require 1290 copies of PepMV CP spanning 510 nm ( 3 . 95 Å of axial rise/subunit ) , which agrees with the 509 nm length originally reported for the virus ( Jones et al . , 1980 ) . The signal clearly separates the individual nucleotides , but we could not identify the bases due to the helical averaging of the variable sequence of the ssRNA . In order to explore the protein–ssRNA interactions , we modeled a polyU and included a set of four PepMV CP monomers in a molecular dynamics flexible fitting ( MDFF ) simulation ( Trabuco et al . , 2008 ) ( Figure 2—figure supplement 1 and 2 ) . The results show that the RNA resides in a continuous groove with a high electropositive potential ( Figure 2A ) constructed by CPs from consecutive turns of the helix . Local resolution measurements in the cryoEM map suggest a high variability in positions 3 and 4 at the segmented density for the ssRNA ( Figure 2B ) . Several amino acids display potential interactions with the phosphate backbone of the ssRNA . S92 and S94 in one face , and Q203 at the opposite side of the ssRNA might interact at positions 1 and 3 ( Figure 2C ) . Backbone torsion angles of 85° and 130° at these positions allow the binding of the base at position 2 into a deep pocket ( Figure 2D ) . Three polar-charged residues , R124 , D163 , and K196 might establish H-bonding contacts with this RNA base . D163 is in the most conserved region within the genus Potexvirus , and the consensus sequence FDFFD ( Figure 2—figure supplement 3 ) constructs the floor of the RNA binding pocket . This pocket is large enough to accommodate pyrimidines or purines , and the presence of three amino acids ensures the interaction with the RNA regardless of the nucleotide . . 10 . 7554/eLife . 11795 . 010Figure 2 . Interaction between PepMV CP and ssRNA . ( A ) Cut-away rendering of four PepMV CP subunits at consecutive turns of the helix with the ssRNA between them . Molecular surfaces are colored according to their electrostatic potential using a color scale that ranges from -5KT ( red ) to +5KT ( blue ) . ( B ) Structural detail for the density of the ssRNA associated to a single PepMV CP . ssRNA is a polyU model with five nucleotides . S1 and S2 indicate switches along the phosphate backbone . The inset renders the density region for ssRNA according to local resolution measurements ( blue around 4 Å , and red at 6 Å of resolution; full color scale in Figure 1—figure supplement 4 ) . ( C , D ) Focus on local protein–RNA interfaces where amino acids with possible interaction with the RNA are highlighted . ( E ) Trans-complementation assays between PepGFPΔCP and several CP mutants . The images show agroinfiltrated N . benthamiana leaves imaged under UV light to detect the expression of GFP . Data were recorded at 4 and 6 days post inoculation ( dpi ) . ( F ) Western blot analysis of the presence of PepMV CP in preparations of fully assembled virions from trans-complementation assays . PepMV CP , Pepino mosaic virus coat protein . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 01010 . 7554/eLife . 11795 . 011Figure 2—figure supplement 1 . Rendering of the atomic model construction used during MDFF for the analysis of the protein-RNA interfaces . Representation of four PepMV subunits and two molecules of ssRNA obtained after MDFF . The set was chosen to explore protein–protein and protein–RNA interactions . CP1 and CP4 also contain the N-terminal arms from adjacent subunits , whereas CP2 and CP3 lack their N-terminal arms . The set was fitted inside a region of the cryoEM map for PepMV . CP , coat protein; MDFF , molecular dynamics flexible fitting; PepMV , Pepino mosaic virus . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 01110 . 7554/eLife . 11795 . 012Figure 2—figure supplement 2 . Progress during the MDFF run . Progress of the MDFF measured as the improvement of the cross-correlation between the atomic coordinates ( shown in Figure 2—figure supplement 1 ) and the cryoEM map for PepMV along the 40 ns of the MDFF run . MDFF , molecular dynamics flexible fitting . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 01210 . 7554/eLife . 11795 . 013Figure 2—figure supplement 3 . Sequence alignment between CP from several representatives of the genus Potexvirus . The alignment was produced by CLC Main Workbench and indicates the degree of conservation and the consensus sequence . The alignment includes CP sequences from: Pepino mosaic virus ( PepMV ) ( NP_663728 ) , Papaya mosaic virus ( PapMV ) ( P16596 ) ; Bamboo mosaic virus ( BaMV ) ( NP_042587 . 1 ) , Potato aucuba mosaic virus ( PAMV ) ( P37993 ) ; Potato virus X ( PVX ) ( P10468 ) ; Narcissus mosaic virus ( NMV ) ( NP_040782 ) ; White clover mosaic virus ( WClMV ) ( NP_620719 ) ; Lily X virus ( LVX ) ( YP_263307 ) ; Cymbidium mosaic virus ( CymMV ) ( Q00467 ) ; Plantago asiatica mosaic virus ( PlAMV ) ( NP_620840 ) ; and Foxtail mosaic virus ( FoMV ) ( NP_040992 ) . The most conserved region includes D163 in PepMV CP ( consensus sequence FDFFD in the alignment ) , one of the amino acids at the binding pocket for the ssRNA . CP , coat protein . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 013 We tested several PepMV CP mutants in trans-complementation assays ( Sempere et al . , 2011 ) . Here , a PepMV construct that expresses GFP instead of CP ( PepGFPΔCP ) acts as reporter of the complementation by the co-expression of selected CP mutants . The fluorescent signal by GFP allowed following the cell-to-cell movement of the virus . The results ( Figure 2E ) reveal that the three amino acids at the binding pocket for nucleotide at position 2 ( R124 , D163 and K196 ) are required to complement the cell-to-cell movement of the CP-defective PepMV mutant , suggesting a disruption of the CP-RNA interaction needed in virus intercellular transport . Mutant K93A also impairs viral propagation within the inoculated leaf , probably due to structural changes at the loop that contains S92 and S94 . In the case of mutant Q203A , it seems that there is a reduced movement between cells but not full impairment . We also tested the production of fully assembled virions analyzing by Western blot virion preparations from infected leaves ( see 'Materials and methods' ) . Attempts to purify PepMV virions were only fruitful for the wt and for the Q203A mutant ( Figure 2F ) , suggesting the lack of full viral assembly in the rest of PepMV CP mutants . Single amino acid changes appear to impair CP-RNA binding , probably including a cooperative effect between the hundreds of CP copies that build the virions . As described for BaMV ( DiMaio et al . , 2015 ) , the helical assembly of PepMV is mediated by the CP N-terminal arm and C-terminal extension . However , the improved resolution of the current data allowed for additional insights . The N-terminal arm from the Ni subunit interacts with a hydrophobic groove of the Ni-1 subunit and establishes the main side-by-side contact in the helical arrangement ( Figures 1A and 3A ) . A hydrophobic pocket in Ni-1 accommodates part of the N-terminal arm , where F28 from Ni fits ( Figure 3B ) . A similar type of interaction was observed between subunits in the crystal structure of PapMV CP via amino acid F13 ( Yang et al . , 2012 ) , and for BaMV , by residue W41 ( DiMaio et al . , 2015 ) . We tested in vivo the relevance of F28 in the assembly of PepMV by assaying a PepMVCP F28A mutant in trans-complementation and viral purification experiments . The F28 mutant allows cell-to-cell movement ( Figure 3C ) but does not produce fully assembled virions ( Figure 3D ) , suggesting that the tested mutation allows for the functional separation of the cell-to-cell movement of PepMV from the encapsidation of fully assembled virions , reinforcing the notion of a non-virion RNP structure during the intercellular movement of potexviruses through plasmodesmata ( Lough et al . , 2000 ) . 10 . 7554/eLife . 11795 . 014Figure 3 . Interactions through N- and C-terminal flexible regions mediate PepMV assembly . ( A ) Ni subunit links to a hydrophobic groove in the Ni-1 subunit via the N-terminal arm . ( B ) In the Ni-1 subunit a pocket of hydrophobic residues allocates F28 from the Ni adjacent subunit . ( C ) Trans-complementation assays show that F28A mutant allows for cell-to-cell movement . ( D ) The analysis by Western blot of virion preparations show no signal for fully assembled virions in the trans-complementation with F28A mutant . PepMVCP mutant K63A is a positive control . ( E ) Six segmented densities for PepMVCP are seen from the inner side of the virion . The subunit Ni is depicted semi-transparent and includes a ribbon representation for the atomic model for PepMV CP . PepMV CP , Pepino mosaic virus coat protein . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 014 On the other hand , the C-terminal extension , a long coil with a short α-helix in its last part , builds the inner wall of the virus ( Figure 1B ) . This C-terminal segment describes a turn that runs along the axial channel and creates a network of small and local interactions ( Figure 3E ) , where each subunit contacts neighbours at upper and lower levels . Thus , the C-terminal extensions are responsible for the axial interactions that support helix formation . As for BaMV ( DiMaio et al . , 2015 ) , the flexible links between PepMV CPs via N-terminal arm and C-terminal region allow for relative movements between CPs and explain the flexuous nature of the virions . All the high-resolution structures for flexuous filamentous plant viruses correspond to representatives of the genus Potexvirus ( family Alphaflexiviridae ) and include our current data and previously reported structures of PapMV CP ( Yang et al . , 2012 ) and BaMV virions ( DiMaio et al . , 2015 ) . We cannot confirm whether members of other families of flexuous filamentous viruses ( Betaflexiviridae , Closteroviridae and Potyviridae ) have a similar fold in their CPs . We have , nevertheless , looked for structural neighbours within the database of Dali server ( Holm and Rosenstrom , 2010 ) . The search revealed that the CP structure from potexviruses shares its topology with the NP of several representatives of genus Phlebovirus ( family Bunyaviridae ) , such as Rift Valley fever virus ( RVFV ) , Toscana virus , and Severe fever with thrombocytopenia syndrome virus ( SFTSV ) . These viruses infect animals , including humans , are transmitted by arthropod vectors , and pose serious public health concerns . Genera Potexvirus and Phlebovirus belong to different superfamilies of RNA viruses when these are classified based on phylogenetic relationships among RNA-dependent RNA polymerases ( RdRp ) and also have different genomic organizations ( Koonin et al . , 2015 ) . Moreover , the organization of phlebovirus particles is very different . They have segmented ( - ) ssRNA associated with the NP in loose RNPs ( Raymond et al . , 2012 ) , and these are protected inside an icosahedral shell of glycoproteins inserted in a membrane , as has been observed for RVFV ( Huiskonen et al . , 2009 , Freiberg et al . , 2008 ) . The topology of CP from potexviruses and of the NP from phleboviruses ( illustrated for RVFV ) are , nonetheless , very similar ( Figures 4A and 4C ) and show a TM-score of 0 . 51 when aligned , pointing to a common family fold ( Zhang and Skolnick , 2005 ) . The similarities include: the overall topology of the all-alpha helical domain; the N-terminal arm and its binding site from the adjacent protomer , and hence , the side-by-side mechanism for oligomerization; the groove for the ssRNA binding; and the relative positions of all these elements . The crystallographic studies with phlebovirus NPs have shown tetrameric , pentameric , and hexameric oligomers , in which each NP interacts with the core of the adjacent subunit via an N-terminal arm ( Zhou et al . , 2013 ) following the same side-by-side polymerization mechanism shown in potexviruses . The divergence between potexvirus CP and phlebovirus NP is in their C-terminal regions . The C-terminus in PepMV CP protrudes from the core domain ( Figure 4B ) and allows longitudinal interactions for helix building . The C-terminus of the phlebovirus NP folds back into the core region and participates in the RNA-binding site that incorporates seven bases/subunit ( Figure 4D ) . The location of the C-terminal arm in phlebovirus NPs cannot support a longitudinal assembly , and hence , their RNPs remain more loosely associated ( Raymond et al . , 2012 ) than in their plant-virus counterparts . 10 . 7554/eLife . 11795 . 015Figure 4 . Structural homology between PepMV CP and NP from phleboviruses . ( A–D ) The atomic structures for the modeled PepMV CP and for the NP from RVFV ( pdb code 4H5O ( Raymond et al . , 2012 ) are depicted in similar orientations . The representations include the respective ssRNAs . Both proteins are colored in rainbow mode and their similar topology is clear when their C-terminal regions are removed ( A and C ) . Their C-termini are seen in grey color for comparison ( B and D ) . PepMV CP , Pepino mosaic virus coat protein; NP , nucleoprotein . DOI: http://dx . doi . org/10 . 7554/eLife . 11795 . 015 Both potexvirus CPs and phlebovirus NPs have in common that they bind and protect the genomic viral ssRNA . In other regards , however , these two groups are different and evolutionary distant , no one would have anticipated the structural homology that we have found . The current work provides data that suggest a horizontal gene transfer event between genera Potexvirus and Phlebovirus , or evolutionarily related forms . While potexvirus and the rest of flexible filamentous viruses infect plants , members of the genus Phlebovirus infect animals . Some representatives of the family Bunyaviridae , toposviruses , are transmitted by and replicate in arthropods , and infect plants . A similar type of virus could have mediated the transfer of CP/NP genes between distant groups of viruses with different host range . Whether the rest of flexible filamentous plant viruses share these genes remains an open question .
Virus preparations were obtained from N . benthamiana plants infected by PepMV-Sp13 ( Aguilar et al . , 2002 ) . For inoculations , carborundum-dusted leaves of 3 weeks old N . benthamiana plants were rubbed with a homogenate consisting of dried material from PepMV-Sp13 infected plants ground in 30 mM sodium phosphate pH 8 . 0 . Inoculated plants were kept in a growth chamber ( 16 hr photoperiod , 18ºC/26ºC night/day , respectively ) for 2 additional weeks . Virions were purified from N . benthamiana systemically infected leaves following a previously described method ( AbouHaidar et al . , 1998 ) slightly modified . Briefly , infected leaves were homogenized in a buffer containing 0 . 1 M Tris-citric acid ( pH 8 . 0 ) , 0 . 2% 2-mercaptoethanol and 0 . 01 M sodium thioglycolate . Triton-X-100 was added ( 1% v/v ) to the homogenized tissue and mixed for 15 min with constant stirring at 4°C . Chloroform was added to a final concentration of 25% and mixed for 30 min with constant stirring at 4°C . Then , the mixture was centrifuged for 15 min at 12 , 000 g , and viruses were precipitated from the aqueous phase by adding PEG 6000 to a concentration of 5% ( w/v ) . The mixture was then kept with constant stirring for 60 min at 4°C , and centrifuged for 15 min at 10 , 000 g . Virus concentration was estimated by OD readings at 260 nm , with ε0 . 1% = 2 . 9 as extinction coefficient ( AbouHaidar et al . , 1998 ) . Virions were kept refrigerated ( 4–6°C ) in suspension buffer ( 0 . 1 M Tris-citric acid pH 8 ) until observation . The PepMVCP gene was cloned into the binary vector pGWB2 giving raise to plasmid pGWB2-CP , and mutants were constructed based on this plasmid using standard overlapping PCR and molecular cloning methods ( Sambrook and Russell , 2001 ) . For functional analysis , we tested the complementation of a PepMV construct ( pBPepGFPΔCP ) that expressed GFP instead of CP by trans CP expression of selected mutants ( Sempere et al . , 2011 ) . Agrobacterium tumefaciens strain C58C1 was transformed with the different CP constructs and used in trans-complementation assays . A . tumefaciens overnight cultures ( 150 ml ) were centrifuged at 2500 g for 10 min , and pellets were resuspended in agroinfiltration buffer ( 10 mM MES pH 5 . 5 , 10 mM MgCl2 , and 100 μM acetosyringone ) until OD600 = 0 . 6 . The resulting suspensions , carrying plasmids pBPepGFPΔCP ( Sempere et al . , 2011 ) , the CP constructs and pBP19 , were mixed in a 3:3:2 ratio and 6 weeks old N . benthamiana plants were vacuum-infiltrated with the mixture . At 4 and 6 days post infiltration , plants were observed under UV light ( 365 nm ) using a handheld lamp ( Blak Ray B100-AP lamp , UV products , Upland , CA 91786 , USA ) . CP was detected by Western blotting in virion preparations and in protein extracts from leaves agroinfiltrated with CP constructs and pBP19 . For protein extractions , 100 mg samples of N . benthamiana leaves were ground in 200 μl of protein extraction buffer ( 0 . 1 M Tris , pH 8 . 0 , 0 . 125 mM 2-mercaptoethanol , 200 μM PMSF , 10% glycerol ) . Crude extracts were mixed with 5x loading buffer and separated by SDS–PAGE followed by staining with Coomassie Brilliant Blue or by electrotransfer to nitrocellulose membranes . Blots were probed with polyclonal antibodies raised in rabbits against PepMV CP ( AC Diagnostics , Fayetteville , AR ) followed by detection with anti-rabbit immunoglobulin G ( IgG ) coupled to horseradish peroxidase ( Promega , Fitchburg , WI ) and chemiluminescence ( SuperSignal West Pico Chemiluminescent Substrate , Thermo Scientific ) . At 6–8 days post infiltration , N . benthamiana plants were harvested and the tissue was used to purify PepMV virions as described above . The PepMV samples were applied to Quantifoil R 2/2 holey carbon grids previously coated with a thin layer of carbon . Grids vitrified in a FEI Vitrobot were then transferred to a Titan Krios ( FEI ) electron microscope that was operated at 300 kV . Images were acquired using a Falcon II detector at nominal magnification of 59 , 000 and calibrated magnification of 102 , 967 ( 1 . 36 Å/pixel ) . Movie frames from the detector were recorded at a speed of 17 frames/s during 3 s . The total specimen dose was ~50 e−/Å2 along 51 frames . Beam-induced motion correction was performed at the level of micrographs ( Li et al . , 2013 ) in frames range 2-27 , resulting an accumulative electron dose in the sample of the corrected images of ~25 e−/Å2 . Contrast transfer function parameters were estimated using CTFTILT ( Mindell and Grigorieff , 2003 ) . Selection of helices was performed using EMAN2 ( Tang et al . , 2007 ) . The resulting data set included 833 selected helices that were processed in SPRING ( Desfosses et al . , 2014 ) software-package following a strategy of single-particle based helical reconstruction . Images were CTF corrected by phase-flipping . Global and local search of optimum helical symmetry parameters ( Figure 1—figure supplement 2 ) resulted in helical rise/rotation of 3 . 95 Å/41 . 1° per subunit ( 8 . 76 subunits/turn and 34 . 6 Å of helical pitch ) . The cryoEM images of helices were excised in overlapping segments of 218 Å length . The segmentation of helices was performed using several different step sizes ( from 8 to 40 Å ) , yielding similar results . The final cryoEM map for PepMV contains information from about 170 , 000 asymmetric units . The resolution was estimated using the Fourier Shell Correlation ( FSC ) calculated between fully independent half-sets ( the so-called 'gold standard' ) and 0 . 5/0 . 143 cutoffs in the FSC correspond to 4 . 5/3 . 9 Å resolution ( Figure 1—figure supplement 3 ) . Local resolution variability was also estimated using ResMap ( Kucukelbir et al . , 2014 ) ( Figure 1—figure supplement 4 ) . The cryoEM map was subjected to an enhancement of high frequencies applying a B-factor of -200 1/Å2 and was low-pass filtered to 3 . 9 Å . The initial atomic model for PepMV CP was generated via iTasser ( Zhang , 2008 ) starting with the structure of the CP from PapMV ( Yang et al . , 2012 ) . Segmentation of the cryoEM map and the initial rigid body fitting of the iTasser model's fragment 50-196 was done manually in Chimera ( Pettersen et al . , 2004 ) . The sequence was set on register , and the rest of the structure built using Coot ( Emsley et al . , 2010 ) . The model was improved by iterative cycles of manual model rebuilding . Refmac5 ( Murshudov et al . , 1999 ) was used to refine the model and to correct geometry/stereo-chemistry problems . Non-crystallographic symmetry was used in order to improve interfaces and minimize clashes between adjacent subunits . The MolProbity and clash score statistics ( Chen et al . , 2010 ) were in the top 100th percentile when compared with atomic structures at similar resolution ( Figure 1—figure supplement 6 ) . The geometry of the ssRNA ( modeled as a polyU ) was further improved using the Rossetta Erraser tool ( Chou et al . , 2013 ) . Molecular dynamics simulations were carried out with NAMD 2 . 9 ( Phillips et al . , 2005 ) through the MDFF plug-in ( Trabuco et al . , 2008 ) . Simulations were run with the CHARMM27 force field with CMAP corrections ( Mackerell et al . , 2004 ) in explicit solvent at a gscale of 0 . 3 . Simulation parameters were kept as specified by the MDFF plug-in with the exception of margin ( 2 ) , cutoff ( 12 ) , switchdist ( 10 ) , pairlistdist ( 16 ) , nonbondedFrequency ( 1 ) , and fullElectFrequency ( 1 ) . Simulation used restraints for secondary structure , chirality and cispeptide derived from the initial atomic model . During the first 10 ns of the simulation RNA atoms were first coupled to the density while keeping the protein backbone atoms constrained and vice versa . Following this , RNA and protein heavy atoms were simultaneously fitted into the density for 30 ns . Finally , 10 , 000 steps of energy minimization were performed with a grid scaling of 0 in order to increase the stability of the resulting structure . Electrostatic surface potential was calculated in Delphi ( Li et al . , 2012 ) . The 3D cryoEM map for PepMV and the derived CP atomic model are deposited in the Electron Microscopy Data Bank ( www . emdatabank . org ) and the Protein Data Bank ( www . rcsb . org ) under accession codes EMD-3236 and 5FN1 .
Flexible filamentous viruses include economically important plant pathogens . Their viral particles contain several hundred copies of a helically arrayed coat protein ( CP ) protecting a ( + ) ssRNA . We describe here a structure at 3 . 9 Å resolution , from electron cryomicroscopy , of Pepino mosaic virus ( PepMV ) , a representative of the genus Potexvirus ( family Alphaflexiviridae ) . Our results allow modeling of the CP and its interactions with viral RNA . The overall fold of PepMV CP resembles that of nucleoproteins ( NPs ) from the genus Phlebovirus ( family Bunyaviridae ) , a group of enveloped ( - ) ssRNA viruses . The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions , which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous , helical rod or a loose ribonucleoprotein . The homology suggests gene transfer between eukaryotic ( + ) and ( - ) ssRNA viruses .
A group of “flexible filamentous” viruses can cause serious diseases in a wide variety of crops and other plants . Each virus particle contains a single molecule called ribonucleic acid ( RNA ) , which is protected by hundreds of copies of a coat protein . The RNA and coat proteins are arranged in a helical fashion to make a flexible rod-shaped particle . The flexibility of these viruses makes it difficult to carry out in-depth studies of their three-dimensional structures . As a result , we do not know how the RNA and coat proteins interact to form the structure of each virus particle . Agirrezabala et al . used a technique called cryo-electron microscopy ( or cryoEM for short ) to generate a highly detailed three-dimensional model of a flexible filamentous virus called Pepino Mosaic Virus . Agirezabala et al . ’s findings reveal how the virus particles assemble , and the interactions between the coat protein and the ssRNA . Unexpectedly , the structure of the coat protein from Pepino Mosiac Virus is very similar to the structure of “nucleoproteins” from a group of viruses called the Phleboviruses , which infect animals . This similarity is striking and suggests that the gene that encodes these proteins has been transferred between the two groups of viruses during evolution . A future challenge is to find out whether this similarity extends to other groups of viruses .