High-resolution comparative atomic structures of two Giardiavirus prototypes infecting G. duodenalis parasite

The Giardia lamblia virus (GLV) is a non-enveloped icosahedral dsRNA and endosymbiont virus that infects the zoonotic protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis), which is a pathogen of mammals, including humans. Elucidating the transmission mechanism of GLV is crucial for gaining an in-depth understanding of the virulence of the virus in G. duodenalis. GLV belongs to the family Totiviridae, which infects yeast and protozoa intracellularly; however, it also transmits extracellularly, similar to the phylogenetically, distantly related toti-like viruses that infect multicellular hosts. The GLV capsid structure is extensively involved in the longstanding discussion concerning extracellular transmission in Totiviridae and toti-like viruses. Hence, this study constructed the first high-resolution comparative atomic models of two GLV strains, namely GLV-HP and GLV-CAT, which showed different intracellular localization and virulence phenotypes, using cryogenic electron microscopy single-particle analysis. The atomic models of the GLV capsids presented swapped C-terminal extensions, extra surface loops, and a lack of cap-snatching pockets, similar to those of toti-like viruses. However, their open pores and absence of the extra crown protein resemble those of other yeast and protozoan Totiviridae viruses, demonstrating the essential structures for extracellular cell-to-cell transmission. The structural comparison between GLV-HP and GLV-CAT indicates the first evidence of critical structural motifs for the transmission and virulence of GLV in G. duodenalis.


Introduction
Giardia duodenalis (syn.G. lamblia and G. intestinalis) is a zoonotic intestinal protozoan parasite that infects the upper part of the small intestine of mammals, including humans.G. duodenalis causes giardiasis, a widespread diarrheal disease in humans [1].The pathogen is classified into eight distinct genetic groups or Assemblages (A-H), with human infection almost exclusively associated with Assemblages A and B, whereas giardiasis in animals is due to host-specific Assemblages (C-H).Assemblages A and B also have zoonotic potential, having been isolated from both humans and animals infected with G. duodenalis [2].Transmission of G. duodenalis occurs through the fecal-oral route by accidental ingestion of cysts, the infective environmental resistant stage of the parasite, by direct contact with stools of humans and animals infected with the parasite or by drinking water or eating food (e.g., fresh produce) contaminated with the parasite's cysts.Outbreaks of giardiasis are frequently reported globally [3,4] as well as recently in EU countries in 2018-2019 [5,6].However, infection with G. duodenalis is still considered a neglected disease; thus, effective anti-parasitic drugs are limited [7], vaccines are not yet available [8,9], and treatment failure with nitroimidazoles, the first-line drug for giardiasis, is increasingly reported, with up to 45% of patients not responding to initial treatment [10].
GLV is phylogenetically close to toti-like viruses compared to other Totiviridae viruses, which hints at an understanding of the evolutionary relevance of Totiviridae viruses and totilike viruses [16,20].Almost all Totiviridae viruses infecting unicellular yeast and protists merely adopt intracellular transmission by frequent mating of cell division and cell fusion in yeasts and protists [17,21,22].In contrast, the toti-like viruses infecting multicellular hosts have employed an extracellular phase and likely transmit adjacent cells using membrane
Totiviridae and toti-like viruses encode two open reading frames (ORFs), ORF1 and ORF2, in their genome [15,16].ORF1 expresses a capsid protein (CP) that assembles an icosahedral capsid.Only for the toti-like viruses is the CP further post-translationally processed to form major CP (MCP) and crown protein (CrP) [16,24].These viral CP (or MCP) and CrP are in charge of the infection of their hosts [24,28]; however, their role in infection is still poorly understood.The CP of Totiviridae viruses has another function: snatching the cap RNA structure of the host.The attachment of the 5'-cap structure (7-methylguanosine linked through a reverse 5'-5' triphosphate bridge; 5'-m 7 GpppX) to mRNA is required for effective translation in eukaryotic cells.Many viruses express enzymes, such as RNA triphosphatase, guanylyltransferase, and methyltransferase, to process viral RNA via several catalytic pathways and create the 5'-cap structure; however, some viruses, especially negative sense single-stranded ((-)ss) RNA viruses, use a cap-snatching approach and utilize a short cleaved capped RNA fragment from the host mRNAs [29,30].The invariant His residue of the CP in the yeast Totiviridae viruses binds covalently to the m 7 GpppG cap structure of the host mRNA, thereby decapping the m 7 Gp moiety, which speculates a unique cap-snatching approach of transferring the m 7 Gp to the 5 0 end of the synthesized viral positive sense (+)ssRNA transcripts [31,32].However, for GLV, the transcripts lack the 5'-cap structure, and a cap-independent internal ribosomal entry site (IRES) could promote the translation of the structure [15,33].
ORF2 encodes RNA-dependent RNA polymerase (RdRp), which is expressed in a fused CP-RdRp form by ribosomal frameshifting [15,16].During capsid assembly, one or a few CP-RdRp(s) are incorporated inside the virus particle [34,35].The internal RdRps function for intraparticle genome synthesis is to synthesize nascent (+)ssRNAs that can exit via the pore(s) of the virus capsid [25,36,37].Intraparticle genome synthesis and the RdRp/pore are considered fundamental requirements of non-enveloped icosahedral dsRNA viruses for sequestering the host's antiviral defense system, which could be triggered by viral dsRNAs [25,37].However, the pores of some icosahedral dsRNA viruses that infect multicellular hosts (e.g., toti-like viruses) are obstructed [23,25,38].Considering the unique phylogenetic clade of GLV in evolution, the surface and pore structures of GLV are key to deeply elucidating the infection and intraparticle genome synthesis mechanisms in the transmission steps of Totiviridae and toti-like viruses.
Recently, two subtypes of GLV, namely, GLV-HP and GLV-CAT, have been described in depth [15].These subtypes have been found to infect the G. duodenalis Assemblage A isolate HP-1, of human origin, and CAT1, of cat origin.Despite limited nucleotide and amino acid divergence, GLV-HP and GLV-CAT show different phenotypes when individually infecting the naive G. duodenalis Assemblage A isolate WBC6 [15].GLV-HP tends to form aggregates and accumulate below the trophozoite plasma membrane and inside the cell body, whereas GLV-CAT does not form aggregates, is scattered in the trophozoite, and does not accumulate in the cell body [15].Additionally, chronic infection of GLV-HP hampers parasite growth more markedly than GLV-CAT, indicating that GLV-HP might have features of a pre-lytic virus [15].
The atomic models of the yeasts ScV-L-A and L-BC [35,39], protozoan TVV2 [40], and toti-like virus OmRV [24,25] have recently been determined; however, the structure of GLV is still limited to an intermediate resolution of 6 Å [20].Here, we describe the first high-resolution structure and atomic model of GLV-HP and GLV-CAT virions, demonstrating the conserved and unique structural features of infection and intracellular genome synthesis in GLV and other Totiviridae or toti-like viruses.The comparison between GLV-HP and GLV-CAT also presents potential structural motifs that could greatly modify the transmission mechanisms and virulence of the GLV strains.

Cryo-EM structural determination, capsid geometry, and atomic models
The first high-resolution capsid structure of two GLV subtypes was determined using cryogenic electron microscopy (cryo-EM) single-particle analysis at a resolution of 2.1 Å and 2.6 Å for GLV-HP and GLV-CAT, respectively (Figs 1A, 1H, and S1 and S1 Table ).As confirmed in our observations (Figs 1B, 1I, and S2) [15], the GLV-HP particles tend to aggregate with each other both within giardia parasite cells and in purified samples, whereas the GLV-CAT particles do not exhibit this behavior.The resolutions achieved from the obtained cryo-EM maps are the highest in the Totiviridae virus database to date, which enables us to discuss the capsid structure and CP-CP interactions more precisely.
The GLV capsid comprises 60 copies of CP dimers named

C-terminal extension and CP-CP interactions
In the GLV capsid structure, the CP-CP interactions are mainly mediated by the adjacent interfaces of CP-A and CP-B ( In the ScV-L-BC capsid, only a very short C-terminal extension of the CP-A has been identified [35], and ScV-L-A, LRV-1, and TVV2 have no interlocking C-terminal extension [39][40][41].Considering the C-terminal structure of these viruses, the C-terminal extension of GLV is longer and interlocks adjacent A and B subunits in both directions, at least in GLV-CAT (Fig 2C -2E).This interlocking mechanism in the GLV capsid is similar to that of the toti-like virus OmRV.The C-terminal extension of the OmRV-AK4 strain swaps the orientation on the Ser1603 residue in the A and B subunits to enable interlocking in both the A to B subunits and the B to A subunits [25].In the OmRV-LZ strain, the long C-terminal extension of the B subunit interacts with several neighboring subunits [24].In the C-terminal extension of the GLV-CAT subunits, similar swapping occurs structurally on the Lys902 residue.Other segmented and large icosahedral dsRNA viruses, such as the megabirnavirus and quadrivirus, show an extra-long Cterminal extension to interact with adjacent subunits or several proximal subunits [23,42].
It is interesting why the C-terminal extension is required for GLV, OmRV, and other segmented icosahedral dsRNA viruses.It has been previously hypothesized that the C-terminal extension may be required for the stability of the larger capsid as a consequence of packaging its own longer genome to tolerate the necessary higher interior pressure [23,25,35].The capsid of ScV-L-A, ScV-L-BC, LRV-1, and TVV2 is 35-42 nm in diameter and packages a 4.5-5.5 kbp dsRNA [35,[39][40][41], while that of the toti-like virus OmRV is 42 nm in diameter and packages a 7.5 kbp dsRNA [24,25].The GLV capsid is 45 nm in diameter and packages a 6.2 kbp dsRNA [15], which implies that the interior pressure of the GLV capsid is slightly less than that of the OmRV capsid but more than or similar to those of the ScV-L-A, ScV-L-BC, and TVV2 capsids.Therefore, the length of the GLV C-terminal extension and its contribution to interlocking correspond to the expected interior pressure in Totiviridae and toti-like viruses.The C-terminal extension is also important for the interior localization of RdRp.However, the structure of the fused CP-RdRp has not been determined in the obtained cryo-EM map.

Structural comparison of CPs within GLV, Totiviridae viruses, and other icosahedral dsRNA viruses
The CPs of T = 1 icosahedral dsRNA viruses share a conserved α-helix-rich α+β structural fold, as does GLV CPs (Fig 1 ); thus, their structural alignments can inform both unique and common structural features within these viruses.To search for CP structures that are akin to that of GLV in the latest PDB repository, the atomic model of the GLV CP was queried in the Dali online server, yielding eight similar CP structures of dsRNA viruses belonging to Totiviridae, including the unclassified toti-like virus OmRV, Chrysoviridae, Megabirnaviridae, and Quardriviridae viruses (S5A Fig) .All the obtained atomic models were further aligned using MUSTANG and generated an RMSD-based structure phylogeny of these CPs (S5B Fig) .Both results indicate that the closest CP structure of GLV is that of the toti-like virus OmRV and that it is distantly related to those of the other Totiviridae viruses infecting yeast and protozoa (S5 Fig) .Total RMSD value between GLV and OmRV CP-B is 5.8 Å (Amino acid sequence coverage: 82%, TM-score = 0.65).Genetic phylogenetic analysis has demonstrated that GLV has its own cluster, which is located between Totiviridae and toti-like viruses [16,20].The structural comparison between the CPs of GLV and OmRV CPs provides new insight into the unclarified relationship between Totiviridae and toti-like viruses, which might reflect their acquisition and loss of functional structures on the capsid.
Proceeding with this assumption, we compared the CP structure of yeast and protozoan Totiviridae viruses with the toti-like virus OmRV.The GLV CP presents two extra surface regions on the capsid surface compared with those of yeasts ScV-L-A and ScV-L-BC and protozoan TVV2, which infect intracellularly (Fig 3A ), and the OmRV CP presents similar extra surface regions on the capsid (Fig 3A).These extra surface regions on the OmRV CP have previously been suggested as being unique to the toti-like virus CPs and not exhibited in the yeast ScV-L-A CP [25].These surface regions are composed of extra loops and are located in amino acid residues 156-169 (loop 1), 364-388 (loop 2), 415-443 (loop 3), 527-546 (loop 4), and 565-583 (loop 5) in the GLV CP, and in amino acid residues 874-883 (loop 1), 1076-1095 (loop 2), 1115-1141 (loop 3), and 1541-1551 (loop 4) in the OmRV CP (boxed regions in Figs 3B and S6).One extra surface region is built up with loop 3 in the GLV and OmRV CPs (blue box or (c) in Fig 3B and 3C), whereas the other extra surface region is built with loops 1, 2, 4, and 5 in the GLV CP or loops 1, 2, and 4 in the OmRV CP (orange, green, and yellow boxes or (a), (b) and (d) in Fig 3B and 3C).The secondary-structure-based amino acid sequence alignment presents no obvious sequence identity between the OmRV loops and GLV loops, although loops 1, 2, and 3 are located in topologically and conformationally similar regions (Figs 3C and S6).GLV and OmRV employ an extracellular phase, unlike other Totiviridae viruses, which only have an intracellular transmission mechanism [15,16,27].
The extracellular transmission mechanism of Totiviridae and toti-like viruses is still unclear; however, these surface loops could have profound roles in extracellular cell-to-cell transmission, such as particle release, membrane penetration, and receptor-binding mediated cell entry.The toti-like virus OmRV and IMNV and the megabirnavirus RnMBV1 also possess additional CrP on their surface, which may facilitate extracellular or horizontal cell-to-cell transmission in multicellular hosts [23,24,43].Considering that the GLV capsid does not express and has no extra surface CrP structurally (Fig 1), CrP must be a non-essential factor in cell-to-cell transmission.These findings show agreement with previous results that indicate that inhibiting CrP interaction with the OmRV capsid does not eliminate the infectivity of OmRV [24,28].Since the amino acid sequence of CrP is not predicted to be a cell-penetrating peptide, CrP might only be implicated in receptor-binding and subsequent endocytosis.Further, OmRV particles without CrPs are still infectious to host mosquito cells [34].To fully understand the transmission mechanisms of Totiviridae and toti-like viruses, it is necessary to study these identified extra surface loops.

5-fold pore structure
The Totiviridae viruses have a pore on each 5-fold axis [35,40] or each 2-fold/3-fold axis [44], possibly for importing cellular nucleotide triphosphates (NTPs) to the intrapartically packaged RdRp(s) and for exporting the synthesized viral (+)ssRNA to the cytosol.In Reoviridae viruses, the pore on the T = 1 inner capsid dynamically and structurally synchronizes with transcribing the nascent viral (+)ssRNAs on the interior RdRp in situ [36,[45][46][47].The structural mechanisms of the pores in Totiviridae viruses are seldom described due to a lack of in-situ transcribing structures.Asymmetric reconstructions were attempted to determine the structure of the CP-RdRp; however, its localization within the capsid remained ambiguous (S7 Fig) .Only a few or one RdRp are randomly incorporated into their capsid particles, unlike Reoviridae viruses [35], although a partially spooled genome structure like that of Reoviridae viruses is observed in capsid-subtracted 2D class projections in our GLV structure (S8 Fig) .GLV-HP and GLV-CAT capsid structures exhibit pores on each 5-fold axis with an outer and inner diameter of 10-11 Å and 19-20 Å, respectively (Fig 4), similar to other Totiviridae viruses [25,35,40].At the center of the pore, the surface is positively charged because of the cluster of the 10 lysine residues (five Lys209 and five Lys217 residues) (the outside view is shown in Fig 4).In contrast, the interior side of the 5-fold pore is negatively charged (the inside view is shown in Fig 4).These positive and negative surface properties are constantly observed in other Totiviridae viruses [25,35,40], which implies their essential functions.The dsRNA viruses infecting multicellular hosts possess similar pores composed of two consecutive arginine residues (RR motif); however, the pores are obstructed [23,25,34,38].In toti-like viruses and human picobirnavirus, simple conformational changes on the pore assist them in opening [25,38].
The electropositive capsid surface could have a role in recruiting negatively charged NTPs to the pores, as previously suggested for icosahedral dsRNA viruses [23,25].A capsid of the human immunodeficiency virus (HIV) also has positively charged pores for recruiting deoxynucleotide triphosphates (dNTPs) for its intraparticle genome synthesis [48,49].However, the positively charged residues of the HIV capsid pores are also critical for trapping and utilizing cellular inositol phosphates (IP5 or IP6) during capsid assembly and maturation [50,51].These findings and previous results imply the multifunctionality and importance of the two Lys residues of the GLV pore.

Lack of putative cap-snatching active pocket
In yeast Totiviridae viruses ScV-L-A and ScV-L-BC, a cap-snatching pocket that exhibits the His residue, His154 in ScV-L-A or His156 in ScV-L-BC, is structurally located on similar regions of each respective capsid [35,39] (Fig 5A).Although the active pocket is built with a certain number of amino acid residues, only the invariant His is known to be functional [32].It is speculated that the protozoan TVV2 CP could have its cap-snatching pocket in a structurally similar location to the yeast CPs [40] (Fig 5A ); however, this is still under discussion.In the GLV and OmRV CPs, the cap-snatching pocket and the invariant His residue cannot be found in the position corresponding to that of the yeast ScV-L-A and ScV-L-BC CPs (Fig 5A).Structural alignments of Totiviridae and toti-like viruses revealed that His154 in the ScV-L-A and His156 in the ScV-L-BC CPs are located on the surface region, which is followed by structurally well-conserved helices in the Totiviridae and toti-like viruses' CPs (Fig 5B).Due to the swapped orientation of the secondary structural elements after the structurally conserved helices in TVV2, GLV, and OmRV CPs, their corresponding His residue is not located structurally in the previously reported putative capsnatching pocket (Fig 5B).Other viral methyltransferases, such as flavivirus NS5 protein, and capsnatching complexes, such as influenza virus PA/PB1/PB2 complex, have a cavity formed by aromatic amino acid residues (Trp, Tyr, and Phe).These viral enzymes show a negatively charged active site next to the positively charged cluster [29,30,52].In the atomic model of GLV and OmRV CPs, no such charged surface is evident in the surface area of the His residues (His288, His387, and His444 in GLV or His986 in OmRV) (Fig 5A).Therefore, these observations strongly suggest that the cap-snatching pocket no longer structurally exists in GLV and OmRV CPs and that the capsids of the Totiviridae viruses have lost the cap-snatching function through evolution.This finding is consistent with the lack of a 5'-cap RNA structure in GLV transcripts [15,33].

Structural differences between GLV-HP and GLV-CAT
GLV-HP and GLV-CAT present distinct intracellular localization and extracellular release efficiency when chronically infecting the G. duodenalis isolate WBC6, which could, in turn, reflect their virulence [15].The GLV-HP particles interact with each other (Figs 1B, 1I, and S2), which causes particle aggregation and affects the preferable intracellular localization [15].Such particle-to-particle interactions should be mediated by the surface of the GLV-HP capsid.Therefore, the structural differences between GLV-HP and GLV-CAT CPs are the focus of much research.
Within the GLV-HP and GLV-CAT CP amino acid sequences, 45 amino acid alterations exist in the structurally visible region (Ile70-Val929) (Figs 6A and S9).Although the capsid surface of GLV-HP seems less charged (Fig 4), these amino acid alterations do not dramatically alter the hydrophobicity and charges on the capsid surface.However, apparent structural differences are observed between GLV-HP and GLV-CAT CP-A/CP-B dimers (Figs 6B and S10).The major structural difference of CP-A is the aforementioned absence of C-terminal extension in GLV-HP (Figs 2 and S10), while that of CP-B is a variable loop that consists of Pro316-Ser330 residues, localizing between the interface of CP-B and that of CP-A (Figs 6B and S10).between the intrasubunit and intersubunit interactions by the F320L point mutation largely contributes to driving the observed structural change in the variable loop.The Phe320 or Leu320 residue in the CP-A is located near the 5-fold axis, which might also affect the observed structural variation there (Fig 6A and 6B).This additional CP-CP interaction, which is mediated by Leu320 and the variable loop in GLV-CAT, should make the CPs' network in the capsid more robust.With the more robust C-terminal interlocking in the GLV-CAT capsid (Fig 2 ), the capsid assembly of GLV-CAT is structurally distinct from that of GLV-HP.differential scanning fluorimetry (nanoDSF) thermographs differences in Ti (inflection temperature) values between GLV-HP and GLV-CAT particles, which may reflect their capsid stability in multiple dissociation or unfolding steps (S11 Fig) .It is also another possibility that a lack of intersubunit interactions, which are mediated by the variable loops within the GLV-HP capsid, might instead allow intersubunit interactions between two virions.

Conclusion
The comparative analysis of the first high-resolution atomic model of two GLV prototypes enables us to present structural insights into acquired and lost functional features in the evolution of dsRNA Totiviridae and toti-like viruses.The two Lys residues that compose the GLV pores are critical for understanding intraparticle genome synthesis and particle assembly in GLV and other Totiviridae viruses.The surface loops that only exhibit in the OmRV and the GLV capsid explain an essential requirement for acquiring extracellular cell-to-cell transmission, in particular, virus entry and egress.The comparative analysis of the two GLV prototypes reveals significant conformational differences in the C-terminal extension and the variable loop, which are critical for CP-CP interactions.It is hypothesized that the loose assembly of the CPs in GLV-HP could expose hydrophobic CP-CP interfaces to facilitate particle-particle interactions.This hypothesis is in agreement with observations in other viral models, such as the VLPs formed by VP40, the MCP of (-)ssRNA Ebola virus, for which mutations at a specific loop in the protein N-terminus alter both protein oligomerization and virus egress [53].Interestingly, these structural changes are potentially responsible for the transmission and virulence of the two GLV strains, which will provide strategies to engineer GLV to enhance its pathogenic effects on G. duodenalis.

Parasite culture and viral particle preparation
The G. duodenalis isolates used in this study comprised HP-1 (Assemblage AI) originally isolated from a human patient [54], and a kind gift from Prof. E. Nohynkova ´(Charles University of Prague, Czech Republic), and CAT-1 (Assemblage AI) originally isolated from a cat [55], and a kind gift from Dr. G.S. Visvesvara (Centers for Disease Control and Prevention, Atlanta, Georgia, USA).HP-1 and CAT-1 are chronically infected with the virus strains GLV-HP and GLV-CAT, respectively [15].Trophozoites were routinely grown in axenic, microaerophilic conditions in a TYI-S33 medium, supplemented with 10% (v/v) adult bovine serum (Euroclone S.p.A., Milan, Italy) and 0.05% (v/v) bovine bile (Sigma-Aldrich, Merck Life Science S.r.l., Milan, Italy) at 37˚C in 10 mL screw-cap tubes (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) and sub-cultured when confluence was reached (every 48-72 h).For viral particle purification, 1 L of the trophozoite culture of each isolate was grown in 50 mL screw-cap tubes (Falcon, Thermo Fisher Scientific, Waltham, MA, USA) with a TYI-S33 medium for 60 h.The tubes were chilled on ice to detach trophozoites, and the parasites were harvested by centrifugation at 900 × g for 10 min at 4˚C.Trophozoite pellets were combined and washed twice with cold PBS by centrifugation at 900 × g for 10 min at 4˚C.Trophozoite pellets were resuspended in 8 mL of cold PBS and lysed by sonication (5-6 times for 30 s at 60% power and a 10% duty cycle) with a Sonoplus ultrasonic homogenizer (Bandelin Electronic, Berlin, Germany).The lysate was centrifuged at 10,400 × g (in JA20 centrifuge, Beckman) for 10 min at 4˚C to remove debris.To sediment the viral particles, the supernatant was layered onto a PBS/1.5 M sucrose solution at a sample/sucrose 4:1 ratio and ultracentrifuged at 264,000 × g on an Optima TLA 100.3 ultracentrifuge (Beckman) for 2 h at 4˚C.The virus pellet was resuspended with 8 mL of cold PBS, CsCl was added at a final density of 1.39 g/mL, and the volume was adjusted to 12 mL with a PBS/CsCl solution at the same density.Virions were banded by density gradient centrifugation at 152,000 x g for 16 h at 4˚C in a SW41 rotor (Beckman Coulter SRL, Milan, Italy).The gradient was then fractionated, and virion-positive fractions were identified by the phenol extraction of nucleic acids, as previously described [15].The positive fractions were pooled and dialyzed overnight in sterile PBS/glycerol 20% (v/v) and stored at -80˚C until use.The overall quality of the prepared purified virions was examined by transmission electron microscopy (TEM), as previously described [15].For the cryo-EM grid preparation, the purified GLV-HP and GLV-CAT particles were further concentrated to 20-30 μL (approximately 10 9 -10 10 particles/mL).alignments, all-to-all RMSD values were calculated in the superimposed structures.These RMSD values served as a distant matrix to generate a structural phylogeny of the viral CPs using a neighbor-joining method in MEGA X [63], as already described earlier [64,65].The aligned structures were also visualized to discover conserved, acquired, and/or lost unique structures in the GLV CP.To calculate the local RMSD, the local_rms command was applied for the aligned GLV-HP and GLV-CAT CP dimers using the PyMOL script collection (PSICO) module [66].

Fig 1 .
Fig 1. Atomic model of capsid and CPs in GLV-HP and GLV-CAT.The T = 1 capsid geometry and CP-A and CP-B organization in (A) GLV-HP and (H) GLV-CAT.Raw cryo-EM micrographs of (B) GLV-HP and (I) GLV-CAT.The close-up views of N-termini and C-termini in CP-A and CP-B are shown in (C-G) for GLV-HP and (J-N) for GLV-CAT.https://doi.org/10.1371/journal.ppat.1012140.g001

Fig 1 )
and the short C-terminal extension that interlocks CP-A and CP-B on the interior side (Fig 2).The C-terminus of CP orients differently in CP-A and CP-B due to the interlocking between two adjacent subunits.Hence, this C-terminal interlocking is identified in two directions in the GLV-CAT capsid: CP-A to CP-B and CP-B to CP-A (Fig 2C-2E).However, only the CP-B to CP-A direction is identified in the GLV-HP capsid (Fig 2A and 2B).This C-terminal interlocking includes three possible hydrogen bonds: Arg671-Asp922, Arg675-Asp928, and Lys774-Asp927 in CP-B to CP-A in GLV-HP (Fig 2B).The C-terminal interlocking includes the same amino acid pairs as Arg671-Asp922 and Lys774-Asp927; however, more intersubunit interactions are identified in GLV-CAT (Fig 2E).In the CP-A to CP-B direction, only two possible hydrogen bonds-Arg675-Asp928 and E872-N917-are present in GLV-CAT (Fig 2D).

Fig 2 .
Fig 2. C-terminal extension structure of the CP-CP interface in the GLV-HP and GLV-CAT capsids.The atomic model of the 5-fold CP complex of (A) GLV-HP (CP-A in green; CP-B in orange) and (C) GLV-CAT (CP-A in light blue; CP-B in pink) is shown from their inside view.The interlocking C-terminal extensions from CP-A to CP-B and those from CP-B to CP-A are highlighted in the surface representation.The close-up views of the interactions between the C-terminal extension and the adjacent capsid are shown in (B) for GLV-HP and in (D) and (E) for GLV-CAT.The amino acid residues involved in the interactions are indicated.The red dashed lines between the amino acid residues indicate predicted hydrogen bonds with their distances in Ångstro ¨m.https://doi.org/10.1371/journal.ppat.1012140.g002

Fig 3 .
Fig 3. Structural comparison of the GLV CP with those of Totiviridae and toti-like viruses.ScV-L-A, ScV-L-BC, and TVV2 CPs are shown in grey.The GLV and OmRV CPs are shown in orange and blue.The unique surface loops of the GLV and the OmRV CPs are shown as transparent surface representation.(A) The CP of GLV and OmRV that infects extracellularly is aligned with that of ScV-L-A, ScV-L-BC, and TVV2, which infect intracellularly.(B) Aligned CP structure of GLV and OmRV.The colored boxes, also shown as (a), (b), (c), and (d), indicate the surface regions of the extra loops that are identified in the GLV and OmRV CPs.(C) Close-up views of the extra loops.The box colors and regions correspond to those in (B).CP-B subunit of GLV-HP (PDB ID: 8R0F), OmRV-LZ (PDB ID: 7D0K), ScV-L-A (PDB ID: 1M1C), ScV-L-BC (PDB ID: 7QWX), and TVV2 (PDB ID: 7LWY) were utilized for the structural alignment.https://doi.org/10.1371/journal.ppat.1012140.g003

Fig 4 .
Fig 4. Pore structure of the GLV capsid.A surface electrostatic potential map of the 5-fold CP complex of GLV-HP and GLV-CAT from outside and inside views is shown in red (negatively charged) and blue (positively charged) scales.A close-up view and cross-section of the pore structure and surface charges are also shown at the bottom.https://doi.org/10.1371/journal.ppat.1012140.g004 Both CP-A and CP-B show high RMSD in the proximal to the 5-fold axis of the capsid (Figs 6B and S10).Notably, Phe320 in GLV-HP forms intrasubunit interactions with Val184, Leu590, and Asn592 in the CP-B (Fig 6C); however, in GLV-CAT, the corresponding Leu320 in CP-B interacts with Asn112 and Gln282 residues in the adjacent CP-A (Fig 6D).The switching

Fig 5 .
Fig 5. Lack of putative cap-snatching pockets in GLV and OmRV.(A) Electrostatic surfaces of Totiviridae and totilike virus CPs are shown in blue (positive) and red (negative) scales.The CP of the yeast Totiviridae viruses ScV-L-A and ScV-L-BC have cap-snatching active pockets and invariant His residues (His154 or His156) (yellow dotted circles).The CP of TVV2 is speculated to have a cap-snatching pocket with three putative His residues (His537, His648, and His658) in a position similar to that in the yeast Totiviridae viruses (yellow dotted circle).However, in the GLV and OmRV CPs, no His residue is observed on the corresponding cap-snatching pocket, although three His residues (His288, His387, His444) in the GLV CP and one His residue (His986) in the OmRV CP are exhibited on the surfaces.(B) Conserved helices and invariant His residue in Totiviridiae and toti-like viruses.A conserved helix-turn-helix and a long helix are highlighted as dotted circles and rectangles in each CP structure.https://doi.org/10.1371/journal.ppat.1012140.g005

Fig 6 .
Fig 6.Amino acid alterations and structural variability of CPs between GLV-HP and GLV-CAT.(A) Amino acid alterations between GLV-HP and GLV-CAT are mapped on a GLV-HP CP-A/CP-B dimer.(B) Local RMSD values are calculated per amino acid residue.The RMSD values are scaled by blue (low RMSD) and red (high RMSD).The yellow pentagon indicates the 5-fold axis of the GLV capsid.(C) Intra-subunit interaction of Phe230 of CP-B in the GLV-HP capsid.(D) Inter-subunit interaction between CP-A and Leu320 of CP-B in the GLV-CAT capsid.https://doi.org/10.1371/journal.ppat.1012140.g006