Zn 2+ -dependent association of cysteine-rich protein with virion orchestrates morphogenesis of rod-shaped viruses

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γ b directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γ b predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γ b binds to the surface of rod-shaped BSMV virions in a Zn 2+ -dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn 2+ -dependent physical association of γ b with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus ) and Benyviridae . Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus

Besides the structural protein CP and nucleic acids, some viral nonstructural proteins, host factors, and small molecules are also required to virion assembly and disassembly [7,8].Plum pox virus (PPV) HC-Pro and P3 proteins are essential for the formation of stable virions [15,16], potato virus X (PVX) TGB1 binds to one end of its helical particle and triggers the disassembly into a translatable form [17,18].Both the CP readthrough proteins of potato moptop virus (PMTV) and beet necrotic yellow vein virus (BNYVV) associate with one extremity of the virus particles, to improve PMTV long-distance movement [19,20] or BNYVV virions assembly [21].Moreover, the cell-to-cell movement of bamboo mosaic virus (BaMV) requires the stable association of virion with the TGB2-and TGB3-based membrane complex [22].As for the host factor, heat shock cognate 70-kDa protein (Hsc70) is physically associated with cucumber necrosis virus (CNV) icosahedral virions to reduce the stability of CNV virions, thereby releasing the encapsidated viral genome to initiate new infection [23].
Metal ions, represented by zinc, also play vital roles in virus packaging, inhibiting the replication of RNA viruses, antiviral immunity, and host-virus interactions [24][25][26].The structural protein Gag precursor of human immunodeficiency virus type 1 (HIV-1) participates in the assembly of budding virion, the nucleocapsid (NC) domain of Gag binds to the 3'-leader of the genomic RNA to initiate the packaging of HIV virions [27].The NC domain or its cleaved version contains two CCHC zinc-finger motifs, mutations of these zinc-finger motifs cause severe defects in viral RNA packaging and infectivity [27,28].Zn 2+ mediates the conformational changes of hepatitis B virus (HBV) CP to increase the production of virus-like particles (VLPs) [29].In plants, the zinc finger domain in tobacco streak virus (TSV) CP is required for the assembly of VLPs, and the stability of TSV particles is significantly increased in the presence of Zn 2+ [30].
Barley stripe mosaic virus (BSMV) is the prototypical member of the genus Hordeivirus (family of Virgaviridae), the rod-shaped virions encapsidated tripartite plus-strand RNAs designated RNAα, RNAβ, and RNAγ [1,31,32].RNAα and RNAγ encoding the viral replicase subunits αa and γa are sufficient to support viral replication.The CP, which is translated directly from the first RNAβ ORF, is associated with viral genomic RNAs (gRNAs) for virion encapsidation.The RNAβ also encodes triple gene block proteins (TGB1, TGB2, and TGB3) for viral cell-to-cell movement.The cryo-electron microscopy structure of BSMV particles at 4.1 Å found two different sizes of virions [33].The wide form virions contain 111 CP subunits per helix period, while the helical parameters of narrow virions are 106, suggesting that the conformation of CP subunits and the interaction with genomic RNAs are different in the two forms.A series of structural data show that the virions of narrow form are more stable than the wide versions due to the stronger inter-subunit contact between different CP subunits or with viral RNAs [3,33].However, mechanisms governing the regulation of wide and narrow particles remain to be elucidated, and whether other factors and/or small molecules exist on the virus particles and function in BSMV morphogenesis remains to be determined.
The 17 kDa cysteine-rich γb protein encoded by BSMV RNAγ has a versatile role in viral replication [34], movement [35], and interactions with distinct host factors [32,[36][37][38][39].In this study, we found that γb specifically binds to the purified rod-shaped BSMV particles in a Zn 2 + -dependent manner.The association of γb with CP is required for the stability and morphogenesis of BSMV virions.In addition, we also provide evidence that the physical association of the CRPs with the rod-shaped virions is a general feature among the members in the families Virgaviridae and Benyviridae.These results reveal a hitherto unknown role of CRPs during viral infection cycle and deepen our understanding of the molecular mechanism underlying virus morphogenesis.

γb is associated with BSMV virions
To identify the host proteins associated with BSMV virions, virus particles were purified from systemically infected leaves of Nicotiana benthamiana using a similar strategy to one described previously [23].Mock-inoculated plants served as negative controls.The purified BSMV virions and the products purified from mock-inoculated plants were separated by 12.5% SDS-PAGE gel, followed by silver staining.The visible gel bands that were present in the purified virions but absent in the negative control were cut and divided into four groups (S1 Fig) , followed by Q-Exactive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.In line with our expectations, we identified a large number of host factors that could be associated with BSMV virions.Surprisingly, the MS results showed that γb proteins were detected in all four groups (S1 Table ).Previously, we found that CPs co-precipitated with γb-3xFlag proteins in the IP products from BSMV γb-3xFlag -infected N. benthamiana leaves [35].These results suggest that γb may be associated with CP in vivo in the context of BSMV infection.
To determine whether γb directly binds to BSMV virions, immunogold labeling experiments were performed (Fig 1A and 1B).Purified BSMV virions were adsorbed onto Formvar/ carbon-coated nickel grids, followed by incubation with γb antibody or BL buffer.After labeling by goat anti-rabbit secondary antibody conjugated to 10 nm colloidal gold particles, the samples were observed by transmission electron microscopy (TEM).The results showed that approximately 50.87% of virions were specifically labeled with gold particles on the surface; in contrast, only a few gold particles were associated with the virions incubated with BL buffer or the pre-immune rabbit serum (Figs 1A and 1B and S2A), and we conducted immunogold labeling experiments with BSMV γb polyclonal antibody in the purified viral particles of lychnis ringspot virus (LRSV; genus Hordeivirus), poa semilatent virus (PSLV; genus Hordeivirus), tobacco rattle virus (TRV; genus Tobravirus), tobacco mosaic virus (TMV; genus Tobamovirus), and BSMV simultaneously.Notely, only the BSMV virions exhibited abundant binding with gold particles (S2B Fig) .These results indicate that γb is associated with the outer surface of BSMV virions.
To clarify the specificity of γb antiserum, we tested the cross-reactivity of BSMV CP protein with the γb antiserum by using BSMV-infected N. benthamiana leaf samples or its mutant BSMV mγb (an AUG!UUG substitution of the γb initiation codon), as well as GST-CP and GST-γb proteins [35], the results demonstrated that the γb antiserum is highly specific and does not cross-react with the CP protein (Fig 1C and 1D).
Altogether, these results indicate that γb interacts with and locates to the surface of BSMV virions.

γb interacts with CP in vivo and in vitro
To test whether the association of γb with BSMV virions depends on the CP-γb interaction, we performed a BSMV-based biomolecular fluorescence complementation (BiFC) assay [35].Recombinant yellow fluorescent protein (YFP) signals were observed at the periphery of chloroplasts in N. benthamiana epidermal cells co-expressing either CP-YFPn with BSMV γb-YFPc or CP-YFPc with BSMV γb-YFPn (Figs 2A and S3).The yeast expressing AD-CP and BD-γb grew well on SD/-Trp-Leu-His-Ade drop-out plates (Fig 2B ), indicating that CP interacts with γb in Y2H system.The GST pull-down result showed that both GST-γb and GST-γb 1-85 directly pulled down CP-His, indicating that the N-terminal of γb (aa 1-85) is sufficient for its interaction with CP in vitro (Fig 2C).
To further confirm the interaction between CP and γb further, we performed microscale thermophoresis (MST) measurements.The concentration of γb-GFP-His protein was kept constant at 10 μM, and different amounts of CP-His protein were titrated starting from Taken together, these results showed that γb protein directly interacts with CP in vivo and in vitro, and the N-terminus of γb is key motif that interacting with CP protein.

γb interacts with CP in a Zn 2+ -dependent manner
Previous studies have shown that the N-terminal (aa 1-85) of γb contains three zinc-binding motifs [40], but the function of the zinc-binding motifs remains largely unknown.Since the N-terminal of γb interacts with CP (Fig 2C ), we hypothesized that the zinc-binding activity may be functionally linked to the γb-CP interaction.The MST assay demonstrated the Zn 2 + -binding activity of γb, but it did not bind to Mg 2+ (Fig 3A).Next, MST, co-IP, and GST pulldown assays were performed to identify the effect of Zn 2+ on the γb-CP interaction, all these results showed that the binding affinity of γb and CP was enhanced by the addition of Zn 2+ in vitro and in vivo (Fig 3B -3D).In contrast, the divalent cation chelator EDTA substantially impaired the CP-γb interaction in the presence of 200 μM Zn 2+ (Fig 3C and 3D).Moreover, the addition of Zn 2+ and Cu 2+ increased the interaction between CP and γb, whereas other bivalent ions had no effect (Fig 3E).Together, these results suggest a positive regulatory role of Zn 2+ in the CP-γb interaction.

γb is physically associated with virions in a Zn 2+ -dependent manner
The majority of viruses within the family Virgaviridae (excluding genus Tobamovirus) encode cysteine-rich proteins (CRPs) [1,31,32].BSMV γb protein consists of 11 cysteines, and mutations of the cysteines at positions 60, 64, 71, and 81 as well as a histidine at position 85 in the C2 region (aa 60-85) of γb, result in the loss of its zinc-binding activity [40].Although CRPs from the genera Hordei-, Peclu-, Gora-, Furo-, and Tobravirus have low sequence similarity, the CCCH-type zinc-binding motifs (Cys-50, Cys-60, Cys-81, and His-85) are highly conserved (Figs 4A and S4).The alanine substitution at His-85 of γb (γb H85A ) mutant, which -binding activity of γb by adding 200 μM EDTA, the interaction between γb and CP was reduced by about fivefold; however, the γb H85A mutant showed the same weak band as γb in the EDTA-treated samples (Fig 4D).These results suggest that the interaction between γb and CP can be enhanced in the presence of Zn 2+ .
To determine whether CP has zinc-binding activity, the amino acid sequences of CP from different viruses were aligned via the Uniprot website (https://www.uniprot.org/),and the potential zinc-binding sites were predicted by using the ZincBinder online server (http://www.proteininformatics.org/mkumar/znbinder)(S5 Fig) .The results showed that the histidine at position 13 (His-13) of CP is a potential zinc binding residue among hordei-, peclu-, and The interaction between γb and CP was served as a positive control.Combinations containing the empty vector pGADT7 or pGBKT7 were used as negative controls.Yeast cells containing the indicated plasmids in the bottom panel were spotted on synthetic dextrose dropout medium, SD/-Trp-Leu or SD/-Trp-Leu-His-Ade. (D and E) His-85 of γb and His-13 of CP are required for zinc binding activity and interaction between CP and γb.γb and γb H85A were fused to GSTtag and incubated with CP-His or CP H13A -His.200 μM EDTA was used to destroy zinc-binding activity in the GST pull-down assays.GFP-His was used as a negative control.Western blot analysis of immunoprecipitated proteins was performed using anti-GST or anti-His antibodies.(F) Immunogold labeling experiments showed that γb binds to BSMV virions by interacting with CP in a Zn 2+ -dependent manner.BSMV and its derivatives virions were purified from infected leaves of N. benthamiana at 5 dpi.Virions were adsorbed onto 200-mesh nickel grids and incubated with antibodies against the γb protein.S6).To investigate the spatial distribution of CP His-13 on virus particles, three-dimensional models of BSMV virions were built based on the reported near-atomic structure of BSMV [33], and the result showed that CP His-13 was located on the virion surface (S7 Fig) , supporting the prediction that His-13 is critical for CP to bind Zn 2+ .Next, to investigate whether CP His-13 is critical for the interaction between γb and CP, GST pull-down was performed by using CP H13A , and the results showed that GST-γb barely pulled down CP H13A -His compared to CP-His.As expected, when the redundant EDTA was present, the interaction of γb with CP was reduced to a similar extent compared to CP H13A (Fig 4E).Altogether, these results indicate the important role of Zn 2+ in the CP-γb interaction.
Based on these results, we hypothesized that cysteine-rich γb protein might bind to rodshaped BSMV virions with the assistance of Zn 2+ .To this end, γb His-85 and CP His-13 were substituted with alanine in BSMV infectious cDNA clone to generate BSMV H85A and BSMV CP-H13A mutants, respectively.Immunogold labeling experiments showed that only a few gold particles were detected on the surface of purified BSMV H85A or BSMV CP-H13A virions compared to the wild-type BSMV (Fig 4F and 4G).On TEM observation, the number of virions per visual field in BSMV H85A and BSMV mγb mutants was much less than in wild-type BSMV.To evaluate the effects of γb on the stability of BSMV virions, γb-3xFlag protein and γb H85A -3xFlag were transiently overexpressed in BSMV mγb -infected N. benthamiana, and the virions of different BSMV derivatives were purified.The TEM results demonstrated that γb, but not γb H85A , could restore the number of BSMV mγb virions to the wild-type level (Fig 4H).Collectively, these results indicate that γb physically associates with virions by interacting with CP in a Zn 2+ -dependent manner.
γb plays a role in BSMV virions assembly γb serves as a replication enhancer [34] and facilitates the assembly of the viral movement complex at chloroplasts [35] Furthermore, to investigate whether the localization of CP-γb complexes correlates with BSMV genomic RNA, CP-GFP and γb-RFP were co-expressed with the plus-sense BSMV Pumilio system [34].The results revealed that CP-GFP and γb-RFP were co-localized with BSMV genomic RNA at the periphery of chloroplasts (Fig 5A ), implying that chloroplasts may be the location where the assembly of BSMV virions occurs.
To further determine whether γb participates in virion assembly, BSMV, BSMV mγb , and BSMV H85A were agroinfiltrated into N. benthamiana leaves, and native extracts were subjected to sucrose gradient centrifugation at 4 days post-infiltration (dpi).Consistent with previous studies [15], the top layer (fractions 1 to 3) were free CP and small-volume CP complexes, while the middle layer (fractions 4 to 7) corresponded to BSMV VLPs, which was further confirmed by TEM (Fig 5B).The results show a substantially higher proportion of CP proteins in the middle layer in BSMV-infected samples.However, in the absence of γb protein expression (BSMVmγb) or when the γb-CP interaction was disrupted in the BSMV H85A mutant, the majority of CP proteins were detected in the top layer (Fig 5B).Altogether, these results suggest that γb promotes the formation of BSMV VLPs.
Given the apparent co-localization of γb and CP in close proximity to the chloroplasts which are the major sites for BSMV replication and contain abundant viral progeny RNAs [34], to determine whether γb promotes BSMV virion assembly by enhancing the RNA-binding activity of CP, we performed an EMSA assay.Since the tripartite genomic RNAs of BSMV shared a highly conserved 3'-UTR, the 300 bp RNA at 3'-UTR labeled with Andy Fluor-647 was used as probe [31,34,35].A specific band appeared when the CP-His protein was included, indicating that CP specifically binds to this RNA probe (Fig 5C).Intriguingly, γb, but not γb H85A , could significantly enhance CP RNA-binding activity (Fig 5C ).Taken together, these results show that γb protein is required for virion assembly by promoting CP RNA-binding capacity.
Previous study has shown that BSMV infection induce cytoplasmic invaginations (CIs) on the chloroplast in BSMV-infected N. benthamiana leaf cells [41].To further observe virion assembly in BSMV-infected cells, the agroinfiltrated N. benthamiana leaves were harvested at 3 dpi, followed by chemical fixing and embedding in resin for immunogold labeling analysis.The results reveal that large quantities of VLPs in CIs of the abnormal chloroplast were labeled with gold particles conjugated to CP antibodies in BSMV-infected leaves.Moreover, gold particles conjugated to γb protein also bound specifically to the surface of these VLPs in vivo (Fig 5D).These results indicate that γb protein tightly binds to BSMV virions in vivo and may coordinate encapsidation at virion assembly sites.Beyond that, we provide persuasive evidence that beyond BSMV replication sites, BSMV virion assembly also occurs at chloroplasts.The close

γb with zinc-binding activity is required for the stability of BSMV virions
Preliminary data suggest that γb may also stabilize BSMV viral particles, which could play dual roles in BSMV morphogenesis (Fig 4H).To clarify this, purified virions were incubated with 1 nM RNaseA at 37˚C, and the result shows that the degradation rate of BSMV mγb genomic RNAs was significantly higher than that of the BSMV (Fig 6A and 6B).Next, Agrobacteria containing BSMV, BSMV mCP , BSMV CP-H13A , BSMV mγb , or BSMV H85A infectious clone were individually infiltrated into N. benthamiana, infiltrated leaf tissues were harvested at 4 dpi and ground in liquid nitrogen, crude extracts were incubated at 37˚C followed by in vivo endogenous RNase sensitivity assay [42,43].Northern blot results show that BSMV genomic RNA was quite stable, owing to the protection by intact particles.However, the viral genome of BSMV CP-H13A and BSMV H85A were degraded quickly, consistent with BSMV mγb and BSMV mCP mutants (Fig 6C and 6D).These results indicate that γb protein can stabilize BSMV particles in vivo and in vitro, coordinating with CP to encapsulate the viral genome and protect it from degradation.

PLOS PATHOGENS
Viral CRP orchestrates rod-shaped virus morphogenesis Considering that Zn 2+ tightens the association of γb protein with BSMV virions, we wondered whether Zn 2+ could enhance virion stability by promoting γb binding to virions.To test this, purified BSMV virions were treated with 0.02 mM EDTA, followed by immunogoldlabeling assays with antibodies against the γb protein.The results show that the number of gold particles labeled on the surface of BSMV virus particles was significantly reduced under EDTA treatment (Fig 6E ), and the virion morphology also changed (Fig 6E).To further confirm these results, RNase sensitivity assay was carried out.Purified BSMV virions were incubated in PBS buffer with 0.02 mM EDTA for 12 h, followed by 1 nM RNaseA treatment.The result shows that the EDTA treatment significantly decreased the stability of BSMV virions compared to PBS control (Fig 6F ).Taken together, these results indicate that stability of BSMV virions requires the association of γb protein with virions.

Physical association of the CRPs with the virions is a general feature in rodshaped viruses
Our data demonstrate that γb is associated with the surface of BSMV virions in a Zn 2+ -dependent manner and is involved in virion assembly and stability.Moreover, since the sequence alignment indicated that zinc-binding amino acids are highly conserved in CRPs and CPs of diverse viruses (Figs 4A and 4B and S4 and S6), we wondered whether the physical association of CRPs with virions is a general feature in rod-shaped plant viruses.To test this, LRSV, PSLV, and TRV in the family Virgaviridae [1], and beet necrotic yellow vein virus (BNYVV; genus Benyvirus) in the family Benyviridae [2] were used for further analysis.BiFC assay confirmed the interaction between CRPs and CPs (S9 Fig) , indicating that these CRPs also associate with their corresponding CPs.
To further investigate the relationship between CRPs and CPs, we performed immunogoldlabeling assays.The purified LRSV [44], PSLV [45], TRV [13,46], and BNYVV [47] virions were adsorbed onto Formvar/carbon-coated nickel grids, followed by incubation with the corresponding CRP antibodies, and the samples were observed by TEM.The results show that LRSV γb, PSLV γb, TRV p16, and BNYVV p14 all tended to bind to their respective virions with different binding affinities ( Fig 7).Taken together, these data reveal a new role of CRPs on virions, which might play important roles in virus morphogenesis among different rodshaped viruses.

Discussion
The life cycle of plant viruses includes viral replication, intra-and intercellular movement, and encapsidation; not only the movement, but also the encapsidation are also coupled with viral replication [48][49][50].Most viruses contain small genomes due to the limitation imposed by encapsidation; in order to efficiently infect host plants, viral proteins interact with each other, resulting in a combinatorial interaction network [51][52][53].The CP protein plays a dominant role in the encapsidation process of viral particles, but little is known about how other viral proteins function in virion assembly.We previously demonstrated the important role of multifunctional γb protein in viral replication [34], cell-to-cell movement [35], and replication-to-movement switch [37].In this study, we extended our investigation by demonstrating that γb binds to virions via physical interaction with CP in a Zn 2+ -dependent manner, which is required for virion assembly and stability.These data reveal a mechanism whereby the multifunctional CRP protein γb directly participates in BSMV virion assembly, which revealed a novel role of γb protein beyond its functions in multiple steps of viral infection, including replication, movement, and assembly.
Self-assembly of virions is a complex oligomerization process, which usually occurs along ordered interactions between CP subunits and includes a series of transient assembly intermediates [7].Once assembled, most virions undergo maturation reactions, such as covalent modification or conformational rearrangement, to increase their stability and protect the viral genome from physicochemical attack [54].However, the virion also has a metastable and dynamic structure that undergoes controlled conformational transitions during viral infection and performs critical functions [7,[54][55][56].Unlike other helical plant viruses, BSMV has two versions of virions, one wider and one narrower.The narrower version contains two lateral inter-subunit salt bridges and is more stable, while the wider version has no inter-subunit salt bridges [57].Here, we found that the binding of γb to virions enhanced the assembly and stability of BSMV virions.Whether the γb-CP interaction alters the inter-subunit contact of the CP subunit and mediates the transition from a wider to a narrower version is unclear, and crystal structural evidence of the γb-CP complex may be helpful to address this question.
An increasing body of evidence indicates that viral-encoded multifunctional small proteins and host factors play important roles in the assembly and disassembly of virions.For example, HC-Pro is associated with PPV particles at one end of the virion to enhance the stability and yield of infectious PPV particles [16,58,59].TGB1 has a negative effect on PVX particles by associating with one end of the particles to mediate their disassemble process [17].In addition, host factor Hsc70-2 is physically bound to CNV and necroviruses and is involved in the stability of virions and viral systemic movement [23,43,60].In this study, we found that γb proteins are attached to the surface of rod-shaped BSMV virions.Our findings are completely different from previous findings regarding the binding mode of potyviral HC-Pro [15,16], PVX TGB1 [17], and the CP readthrough proteins of PMTV [19,20] and BNYVV [21], as they are all associated with virus particles at one end of the virion.These findings shed new light on the important role of non-coat proteins in virion assembly and stability.However, whether the association of γb and virions improves the virus performance or even seed transmission efficiency in natural conditions is still an open question.
Intriguingly, CPs from hordei-, gora-and pecluviruses have conserved His-13 at their Nterminal and are distributed on the surface of virions, which is predicted as a zinc binding residue (S6 and S7 Figs).Benyviral CP also contains a His-15 that may be involved in zinc binding activity (S10 Fig) .Interestingly, we found that there was a conserved histidine at the C-terminus of tobraviral CP, and the furoviral CP contained a possible HHCC-type zinc finger motif; pomovirus has two histidines around His-55 and His-140, which may be potential binding sites for zinc ions.In summary, both CPs and CRPs of almost all members of the families Virgaviridae and Benyviridae have potential Zn 2+ binding sites (S10 Fig), thus targeting the CRPs to virions may be a general strategy employed by diverse rod-shaped and filamentous plant viruses for virus morphogenesis.

Plant growth conditions
Growth conditions of N. benthamiana plants and barley (Yangfu 4056) were described previously [37].

Plasmid construction
All constructs described below were validated by DNA sequencing.The corresponding primers used in this study are listed in S2 Table.
For GST pull-down assays, BSMV γb and its derivatives were individually cloned into pGEX-KG vector [63], BSMV CP and its derivatives were cloned into pET30a (+) vector.For MST assay, BSMV γb and its derivatives were individually cloned into pET30a-GFP vector.All the recombinant proteins were purified by using Escherichia coli (BL21 strain).
For co-immunoprecipitation (co-IP) experiments, γb and its derivatives were integrated into SpeI and SacI restriction sites of pMDC32 vector [43].

Virus inoculation
For N. benthamiana leaves, Agrobacterium tumefaciens EHA105 containing pCB301-RNAα, pCB301-RNAβ, and pCB301-RNAγ (or its derivatives) were mixed at equal volumes to a final OD 600 = 0.3, and infiltrated into leaves of 3-4-week-old N. benthamiana plants.For barley leaves, N. benthamiana leaves inoculated with BSMV or its derivatives were harvested at 5-10 dpi and then ground in 10 mM sodium phosphate (pH 7.2) containing 0.5% freshly prepared sodium sulfite, and barley leaves at the 2-leaf stage are inoculated directly with the ground sap of N. benthamiana leaves by mechanical rubbing [66,67].

Virion purification and VLPs isolation
Viral inoculated leaves were collected at 5-10 dpi and ground in liquid nitrogen.10 g N. benthamiana leaf tissue was extracted in 20 mL precooled 0.5 M boric acid buffer (pH 9.0).After filtering with gauze, the homogenate was centrifuged at 8000 g for 10 min at 4˚C. 1/20 volume of the supernatant 20% Triton X-100 was added into the crude extract, and then subjected to ultracentrifugation at 40,000 rpm for 2 h at 4˚C in a Hitachi type P70AT rotor.The pelleted was resuspended in 1.5 mL 50 mM PB (0.1 M KH 2 PO 4 , 0.1M K 2 HPO 4 , pH 6.85), and then add 20% Triton X-100 into a final concentration of 1%, which is subjected to sucrose density gradient centrifugation at 35,000 rpm for 2 h at 14˚C in a Hitachi type P40ST rotor.The pellet was resuspended in 1 mL 10 mM PB, and the supernatant were BSMV virions.The virus concentration was determined spectrophotometrically, the absorbance at 260 nm of a 1 mg/ mL suspension of BSMV is 2.6.
Isolation of BSMV VLPs was performed as described previously with minor modifications [15].Briefly, infiltrated N. benthamiana leaf tissues (250-300 mg) were harvested at 4 dpi, ground in liquid nitrogen and suspended in 200 μL borate buffer.The mixtures were centrifuged at 4˚C for 10 min at 3,000 g, and subjected to centrifugation in continuous sucrose gradients (5 to 40%) of 4.8 mL in borate buffer.The gradients were centrifuged at 4˚C for 20 min at 230000 g in a Hitachi type SW55 rotor.Fractions of approximately 300 μL were collected by capillary gravity from the bottom of the tube via a syringe and subjected to western blot analysis.

Transmission electron microscopy (TEM)
TEM was performed as described previously [41].Purified virions were adsorbed onto 200-mesh nickel grids for 5 min.The samples were stained with 2% uranyl acetate for 1 min in the dark, the grids were then viewed with a Hitachi H-7650 or a JEM-1230 transmission electron microscope operated at 80 kV.

PLOS PATHOGENS
Viral CRP orchestrates rod-shaped virus morphogenesis

Immunogold labeling assay
Immunogold labeling assay was performed as described previously with minor modifications [41].Purified virions were adsorbed onto 200-mesh nickel grids for 5 min, and dry the grids.Then placed the grids on BL buffer [1×PBST (pH 7.5, 0.05% Tween 20), 0.05% Triton X-100, and 1% BSA] for 10 min to reduce nonspecific binding of antibodies.Rabbit anti-γb polyclonal antibody was diluted 1:100 in BL buffer incubated with the nickel grids at room temperature for 2 hours.Buffer without antibody serves as negative control.The grids were washed 6 times with BL buffer for 2 min each.Dry the nickel grids under the heating lamp.The goat anti-rabbit secondary antibody conjugated with 10-nm gold particles (Sigma) was diluted as the ratio 1:100 in BL buffer and incubated for 1 hour at room temperature.After the grids were washed 6 times with BL buffer and 4 times with ddH 2 O for 2 min each, stained with 2% uranyl acetate for 1 min in the dark, dry the nickel grids again under the heating lamp.And then viewed with a Hitachi H-7650 or a JEM-1230 transmission electron microscope operated at 80 kV.The antiserums against BSMV γb and CP, LRSV γb, PSLV γb, TRV p16, and BNYVV p14 were all obtained from rabbits by Beijing Protein Innovation Co., Ltd., the pre-immune serum from rabbit was utilized in the immunogold labeling assay as a negative control.

Microscale thermophoresis (MST)
The Microscale Thermophoresis (MST) assay was performed as described previously with minor modifications [68].γb fused to a GFP-6×His tag was purified from E. coli BL21 strain.The concentration of the γb-GFP-His should yield between 100-1500 fluorescence counts when measured with the Monolith NT.115.After a short incubation of the target protein γb-GFP-His with CP or metal ion ligands in PBS buffer (1xPBS, 0.1% Tween 20), the samples were loaded into Monolith NT.115 Standard Treated capillaries.Measurements were made at 25˚C.And then the resulting data was digitized using the Monolith NT.115 instrument.

Subcellular localization assays and confocal microscopy
Subcellular localization assays were carried out as described previously [37].Co-localization analysis of fluorescence were processed on the pixel-based method with the ImageJ plot profile tool [37].

Yeast two-hybrid (Y2H) assay
The yeast two-hybrid (Y2H) assay were performed as described previously [34].Various γb derivatives were cloned into the pGBKT7 vectors, followed by transformation into the Y2HGold strain.CP derivatives were cloned into the pGADT7 vectors, followed by transformation into the Y187 strain.Mated yeasts were cultured at 30˚C by shaking at 250 rpm for 20 h.The yeast cells were collected and adjusted to an OD 600 = 1.0, followed by gradient dilutions with ddH 2 O, and 2 μL resuspended yeast cells were pipetted onto SD/-Trp-Leu plates and SD/-Trp-Leu-His-Ade plates.The yeast is cultured at 30˚C about 4 days before taking photographs.

Fig 1 .
Fig 1. γb protein is associated with the surface of purified BSMV virions.(A) Representative immunogold labeling images showing γb protein bound to purified BSMV virions (left three photos), the labeling without γb antibody was used as a negative control (right two photos).Representative data are shown and the experiments have done more than three biological replicates with similar results.Scale bar, 200 nm.(B) The average amount of γb proteins associated with purified BSMV virions as shown in Fig 1A.Different letters above the bars indicate statistically significant differences (p < 0.05) as determined by Duncan's multiple range test (n = 10).(C) Western blot to detect the target protein expression in BSMV-infected N. benthamiana leaves with CP and γb antiserum.The BSMV mγb mutant has a UUG to AUG substitution to destroy the translation of γb.(D) Detection of the cross-reactivity between γb and CP antibodies.GST-CP and GST-γb were purified followed by Western blot using CP and γb antibodies.https://doi.org/10.1371/journal.ppat.1012311.g001

Fig 2 .
Fig 2. γb interacts with CP in vivo and in vitro.(A) Interaction of γb with CP in the context of BSMV infection by using BiFC assay.CP-YFPn or CP-YFPc was co-expressed with BSMV γb-YFPn or BSMV γb-YFPc .Chloroplast autofluorescence is shown as false red color.The negative control of BiFC assay is shown in S3 Fig. Scale bars, 20 μm.(B) Yeast two-hybrid (Y2H) assay to analyze the interaction between CP and γb.γb was cloned as a translational fusion with BD, CP was cloned as a translational fusions with AD, and combinations containing empty AD or BD constructs were served as negative control.Serial 10-fold dilutions of liquid cultures were spotted on synthetic dextrose dropout medium, SD/-Trp-Leu or SD/-Trp-Leu-His-Ade. (C) GST pull-down assay for the interaction between CP and γb in vitro.Various γb mutants were fused with GST-tag and incubated with CP-His with or without the 10 μg RNaseA treatment.GST-GFP or GFP-His was used as a negative control.The immunoprecipitated proteins were analyzed by Western blot analysis using anti-GST or anti-His antibodies.(D) Validate the interaction between CP and γb by MST assay.The concentration of γb-GFP-His was held constantly at 10 μM and the concentration of CP-His was titrated from the 20 μM.MST measurements were performed at 25˚C by using 20% LED-power and 40% MST-power.Data analyses were used the MO.Affinity Analysis (x86) software.The dissociation constant (K d ) was derived to be K d = 0.46 μM.https://doi.org/10.1371/journal.ppat.1012311.g002

Fig 3 .
Fig 3.The interaction between γb and CP is specifically enhanced by Zn 2+ in vivo and in vitro.(A) MST assays showed that γb binds to the Zn 2+ .The γb-GFP-His protein was purified from E. coli.In this assay, the concentration of γb-GFP-His was consistent, while 5 μM Zn 2+ or Mg 2+ were used as ligands.The apparent dissociation constant between γb and Zn 2+ obtained was K d = 1.01 μM.The obtained data were digitized using a Monolith NT.115 instrument.(B) MST assays showed that the interaction between γb and CP was enhanced by Zn 2+ .The γb protein alone or the γb protein containing 5 μM Zn 2+ in the buffer was kept constant and the CP protein was added as a ligand.In the absence of Zn 2+ , γb was bound to the CP membrane with K d = 0.63 μM.In the presence of Zn 2+ , the affinity increased 4-fold with K d = 0.15 μM.The resulting data were digitized with Monolith NT.115 instrument.Error bars indicate the range of data points obtained from at least two measurements.(C) Zn 2+ enhances CP-γb interaction by Co-IP assay.N. benthamiana leaf tissues co-agroinfiltrated with RNAα, RNAβ, and RNAγ γb-3xFlag were harvested at 3 dpi.Various concentrations of Zn 2+ were added to the crude protein extracts.Total proteins were immunoprecipitated with anti-Flag beads.Input and immunoprecipitated protein (IP) were analyzed by Western blot analysis with an anti-GFP or anti-Flag antibody.(D) Zn 2+ can enhance the interaction between γb and CP by GST Pull-down assay.Purified GST-γb and CP-His were incubated with a concentration gradient of Zn 2+ from 2 μM to 200 μM.Various concentrations of EDTA were used to destroy the zinc-binding activity.Western blot analysis of immunoprecipitated proteins was performed using anti-GST or anti-His antibodies.(E) Zn 2+ specifically enhances the interaction between γb and CP.The concentration of divalent metal cations (Cu 2+ , Ni 2+ , Mn 2+ , Mg 2+ , Ca 2+ , and Zn 2+ ) was 20 μM.https://doi.org/10.1371/journal.ppat.1012311.g003

Fig 4 .
Fig 4. γb physically associates with virions by interacting with CP in a Zn 2+ -dependent manner.(A) Amino acid sequence alignment of CRP proteins in different genera of Virgaviridae.Sequence alignment was performed by using the Uniprot online server (https://www.uniprot.org/).The conserved CCCH motif (Cys-50, Cys-60, Cys-81 and His-85 for BSMV strain ND18) are highlighted in gray.(B) Amino acid sequence alignment of CPs in different genera of Virgaviridae.The N-terminal of CPs among Virgaviridae were analyzed by using the Uniprot online server.The conserved His 13 of BSMV strain ND18 is highlighted in gray.(C) Yeast two-hybrid (Y2H) assay to analyze the interaction between CP and γb H85A .γb H85A was cloned as a translational fusion with BD.The interaction between γb and CP was served as a positive control.Combinations containing the empty vector pGADT7 or pGBKT7 were used as negative controls.Yeast cells containing the indicated plasmids in the bottom panel were spotted on synthetic dextrose dropout medium, SD/-Trp-Leu or SD/-Trp-Leu-His-Ade. (D and E) His-85 of γb and His-13 of CP are required for zinc binding activity and interaction between CP and γb.γb and γb H85A were fused to GSTtag and incubated with CP-His or CP H13A -His.200 μM EDTA was used to destroy zinc-binding activity in the GST pull-down assays.GFP-His was used as a negative control.Western blot analysis of immunoprecipitated proteins was performed using anti-GST or anti-His antibodies.(F) Immunogold labeling experiments showed that γb binds to BSMV virions by interacting with CP in a Zn 2+ -dependent manner.BSMV and its derivatives virions were purified from infected leaves of N. benthamiana at 5 dpi.Virions were adsorbed onto 200-mesh nickel grids and incubated with antibodies against the γb protein.The pictures were visualized by TEM at 80 kV.Scale bar, 100 nm.(G) Average amounts of γb protein that are associated with the surface of purified BSMV virions as shown in Fig 4F.Different letters above the bars denote statistically significant differences (p < 0.05) determined by the Duncan's multiple range test (n = 13).(H) Quantification of the average number of virus particles.BSMV, BSMV mCP , and BSMV mγb were agroinfiltrated into N. benthamiana leaves.γb-3xFlag or γb H85A -3xFlag was expressed with BSMV mγb infectious cDNA clone.The virions of different derivatives were purified at 5 dpi.Different letters in the chart denote statistically significant differences among different groups according to the Duncan's multiple range test (p < 0.05) (n = 10).
Fig 4. γb physically associates with virions by interacting with CP in a Zn 2+ -dependent manner.(A) Amino acid sequence alignment of CRP proteins in different genera of Virgaviridae.Sequence alignment was performed by using the Uniprot online server (https://www.uniprot.org/).The conserved CCCH motif (Cys-50, Cys-60, Cys-81 and His-85 for BSMV strain ND18) are highlighted in gray.(B) Amino acid sequence alignment of CPs in different genera of Virgaviridae.The N-terminal of CPs among Virgaviridae were analyzed by using the Uniprot online server.The conserved His 13 of BSMV strain ND18 is highlighted in gray.(C) Yeast two-hybrid (Y2H) assay to analyze the interaction between CP and γb H85A .γb H85A was cloned as a translational fusion with BD.The interaction between γb and CP was served as a positive control.Combinations containing the empty vector pGADT7 or pGBKT7 were used as negative controls.Yeast cells containing the indicated plasmids in the bottom panel were spotted on synthetic dextrose dropout medium, SD/-Trp-Leu or SD/-Trp-Leu-His-Ade. (D and E) His-85 of γb and His-13 of CP are required for zinc binding activity and interaction between CP and γb.γb and γb H85A were fused to GSTtag and incubated with CP-His or CP H13A -His.200 μM EDTA was used to destroy zinc-binding activity in the GST pull-down assays.GFP-His was used as a negative control.Western blot analysis of immunoprecipitated proteins was performed using anti-GST or anti-His antibodies.(F) Immunogold labeling experiments showed that γb binds to BSMV virions by interacting with CP in a Zn 2+ -dependent manner.BSMV and its derivatives virions were purified from infected leaves of N. benthamiana at 5 dpi.Virions were adsorbed onto 200-mesh nickel grids and incubated with antibodies against the γb protein.The pictures were visualized by TEM at 80 kV.Scale bar, 100 nm.(G) Average amounts of γb protein that are associated with the surface of purified BSMV virions as shown in Fig 4F.Different letters above the bars denote statistically significant differences (p < 0.05) determined by the Duncan's multiple range test (n = 13).(H) Quantification of the average number of virus particles.BSMV, BSMV mCP , and BSMV mγb were agroinfiltrated into N. benthamiana leaves.γb-3xFlag or γb H85A -3xFlag was expressed with BSMV mγb infectious cDNA clone.The virions of different derivatives were purified at 5 dpi.Different letters in the chart denote statistically significant differences among different groups according to the Duncan's multiple range test (p < 0.05) (n = 10).https://doi.org/10.1371/journal.ppat.1012311.g004 . However, where the BSMV virions assembly occurs remains an open question.CP-GFP alone resulted in fluorescent puncta mainly present in the cytoplasm (S8A Fig), whereas CP-GFP and γb-RFP were co-localized at the periphery of chloroplasts in a movement-deficient BSMV mutant (RNAα + RNAγ)-infected N. benthamiana, which is consistent with the BiFC results in Fig 2A (S8B Fig).

Fig 5 .
Fig 5. γb enhances virion assembly by interacting with CP. (A) Co-localization analyses of γb, CP, and plus-strand BSMV RNAs in RNAα + RNAγ-infected N. benthamiana epidermal cells at 3 dpi.Mixtures of A. tumefaciens containing CP-RFP, CFP-γb, and the split YFP-tagged Pumilio proteins were agroinfiltrated into RNAα and RNAγ (+)PUM -infected leaves [34].Co-localization analyses were visualized by confocal microscopy at 3 dpi.Chloroplast autofluorescence was shown as false pink color.Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP channels along the white dashed line in the merged confocal images.Scale bars, 30 μm. (B) Extracts of BSMV-, BSMV mγb -, and BSMV H85A -infected leaves were analyzed after sucrose gradient centrifugation.Ten fractions were collected from the bottom and the top eight fractions were subjected to CP-specific immunoblot analysis.The right panel indicates a schematic representation of VLPs separation by sucrose gradient centrifugation.The bottom panel indicates the fractions observed under electron microscope.Scale bars, 100 μm.(C) EMSA assay to analyze the effect of γb on the binding between CP and viral RNAs.The 300 bp RNA probe labeled with andy-fluor-647.Unlabeled RNA probe (cold probe) was used as a control.The fusion proteins and probes used for EMSA analyses are indicated on the right of each panel.(D) Immunogold labeling of γb and CP in BSMV-infected N. benthamiana cells show that γb protein binds to VLPs in BSMVinfected leaves.Chl, Chloroplast; CI, cytoplasmic invagination; VLP, virion-like particle.Scale bars, 0.2 μm.https://doi.org/10.1371/journal.ppat.1012311.g005

Fig 6 .
Fig 6. γb is required for the stability of BSMV virions.(A) RNase sensitivity assay in BSMV and BSMV mγb virions extracts.0.2 ng/μL RNaseA were added to the purified BSMV and BSMV mγb virions, and incubated at 37˚C at 0 to 30 min.Viral RNA was extracted followed by Northern blot.(B) Quantification of viral RNA degradation rates in Fig 6A.The relative viral RNA level at 0 min was set to 1.0.Data are shown from three independent repeats; error bars indicate standard deviation (n = 3).(C) Endogenous RNase sensitivity assay in extracts from N. benthamiana infected with BSMV and its derivatives.Inoculated leaves were ground and incubated in PIPES buffer at 37˚C at 0 to 30 min, to allow degradation of unprotected RNA by the endogenous RNase.The total RNAs were then extracted and analyzed by Northern blot.Methylene blue-stained rRNAs served as RNA loading controls.(D) Quantification of viral RNA degradation rates in Fig 6C.The relative viral RNA level at 0 min was set to 1.0.Data are shown from three independent repeats; error bars indicate standard deviation (n = 3).(E) Immunogold labeling experiments show that Zn 2+ can enhance virion stability by promoting γb binding to virions.The purified BSMV virions were treated with 0.02 mM EDTA at 4˚C for 12 h, followed by immunogold labeling assays.The first column (without EDTA treatment) serves as negative control.Average amounts of γb protein that associated with purified BSMV virions with or without EDTA treatment as shown in the right panel.Different letters above the bars denote statistically significant differences (p < 0.05) determined by the Duncan's multiple range test (n = 8).Scale bars, 100 μm.(F) RNase sensitivity assay in extracts of BSMV virions under the EDTA treatment.The purified BSMV virions were treated with 0.02 mM EDTA or PBS buffer and incubated with 1 nM RNaseA at 37˚C at 0 to 30 min.Viral RNA was extracted and detected by Northern blot.https://doi.org/10.1371/journal.ppat.1012311.g006

Fig 7 .
Fig 7. The physical binding of CRPs to the corresponding virions is a general feature among rod-shaped viruses.The virions of LRSV, TRV, and BNYVV were purified from the infected N. benthamiana leaves, the virions of PSLV were purified from infected barley leaves (Yangfu 4056) at 10 dpi.The virions were adsorbed onto 200-mesh nickel grids, followed by incubated with antibodies against the corresponding CRPs or BL buffer (as control) at room temperature for 2 h.The pictures were visualized by TEM at 80 kV.Scale bars, 100 μm.https://doi.org/10.1371/journal.ppat.1012311.g007