Atomic Structure of the Trichomonas vaginalis Double-Stranded RNA Virus 2

Abstract

Trichomonas vaginalis, the causative pathogen for the most common nonviral sexually transmitted infection worldwide, is itself frequently infected with one or more of the four types of small double-stranded RNA (dsRNA) Trichomonas vaginalis viruses (TVV1 to 4, genus Trichomonasvirus, family Totiviridae). Each TVV encloses a nonsegmented genome within a single-layered capsid and replicates entirely intracellularly, like many dsRNA viruses, and unlike those in the Reoviridae family. Here, we have determined the structure of TVV2 by cryo-electron microscopy (cryoEM) at 3.6 Å resolution and derived an atomic model of its capsid. TVV2 has an icosahedral, T = 2*, capsid comprised of 60 copies of the icosahedral asymmetric unit (a dimer of the two capsid shell protein [CSP] conformers, CSP-A and CSP-B), typical of icosahedral dsRNA virus capsids. However, unlike the robust CSP-interlocking interactions such as the use of auxiliary "clamping" proteins among Reoviridae, only lateral CSP interactions are observed in TVV2, consistent with an assembly strategy optimized for TVVs' intracellular-only replication cycles within their protozoan host. The atomic model reveals both a mostly negatively charged capsid interior, which is conducive to movement of the loosely packed genome, and channels at the 5-fold vertices, which we suggest as routes of mRNA release during transcription. Structural comparison of TVV2 to the Saccharomyces cerevisiae L-A virus reveals a conserved helix-rich fold within the CSP and putative guanylyltransferase domain along the capsid exterior, suggesting conserved mRNA maintenance strategies among Totiviridae This first atomic structure of a TVV provides a framework to guide future biochemical investigations into the interplay between Trichomonas vaginalis and its viruses.IMPORTANCETrichomonas vaginalis viruses (TVVs) are double-stranded RNA (dsRNA) viruses that cohabitate in Trichomonas vaginalis, the causative pathogen of trichomoniasis, the most common nonviral sexually transmitted disease worldwide. Featuring an unsegmented dsRNA genome encoding a single capsid shell protein (CSP), TVVs contrast with multisegmented dsRNA viruses, such as the diarrhea-causing rotavirus, whose larger genome is split into 10 dsRNA segments encoding 5 unique capsid proteins. To determine how TVVs incorporate the requisite functionalities for viral replication into their limited proteome, we derived the atomic model of TVV2, a first for TVVs. Our results reveal the intersubunit interactions driving CSP association for capsid assembly and the properties that govern organization and maintenance of the viral genome. Structural comparison between TVV2 capsids and those of distantly related dsRNA viruses indicates conserved strategies of nascent RNA release and a putative viral guanylyltransferase domain implicated in the cytoplasmic maintenance of viral messenger and genomic RNA.

Keywords: Totiviridae; Trichomonas vaginalis; cryoEM; double-stranded RNA virus; subparticle reconstruction.

FIG 1

CryoEM reconstruction and atomic model of TVV2 capsid. (A) 4.0 Å cryoEM map of TVV2 icosahedral viral capsid, radially colored (rainbow) to enhance detail. Icosahedron edges are overlaid to indicate symmetry sites. Two conformers, CSP-A (magenta) and CSP-B (cyan), are displayed as surfaces fit into the cryoEM map. (B) Ribbon diagram of the conformer pair as shown in the cryoEM map, rainbow-colored from blue (N terminus) to red (C terminus). Residues preceding S37 and following P700 (CSP-A) and K701 (CSP-B) are not modeled. (C) Representative sample of the 3.6 Å subparticle map (gray mesh) with side chains of residues 156 to 161 (top, blue), and atomic structure of residues 177 to 196 (bottom, cyan) shown as fit.

FIG 2

Characterization of the capsid shell protein. (A) Surface diagrams of decamers subunits with CSP-As (magenta) nearest the I5 vertex and surrounded by CSP-Bs (cyan) viewed from the exterior (left), side (middle), and interior (right). Approximate measurements of the conformer dimensions are overlaid in Å. Stabilizing protrusions are labeled with black pointers. (B and C) Orthogonal views of A and B conformers (CSP-A and CSP-B), respectively, with secondary structural elements labeled, symbols indicating sites of icosahedral symmetry, and mitten shape provided to enhance visualization. Colored: carapace, green; apical, yellow; dimerization domain, orange. (D) Ribbon diagram of CSP-A (magenta) and CSP-B (cyan) superimposed upon one another. (E) Close-up view of sites of structural discrepancies between conformers display on winding of α4 (top) and α15 (bottom) from CSP-A to CSP-B.

FIG 3

CSP interactions coordinate capsid structure. (A) Six copies of both CSP-A and CSP-B conformers fit into the TVV2 cryoEM map as viewed down the axis of I2 symmetry. Buried surface area (Ų) between subunit interfaces are listed at right. (B) Ribbon diagram of TVV capsid proteins illustrating contact surfaces between two CSP-As (magenta) and 3 CSP-Bs belonging to different decameric units (cyan, green, goldenrod). (C) Closer view of B₁A₁ interface with A₁ and A₃ surfaces displayed to illustrate contacts. (D) Close-up view of the unwound α4 of CSP-B with involved CSP-B residues shown. (E) View of the unwound α15 of CSP-B inserting beneath the long helices of the viral carapace domain. (F) View down 3-fold axis with residues involved in 3-fold symmetry position shown. (G) Opposing β-strands with β17 from both CSPs demonstrating slight twist toward β-sheet like character. (H) Electrostatic potential surface map displaying the positively charged residues of the β15-β16 loop of CSP-B, fit into the negatively charged (red) pocket between carapace and dimerization domains of CSP-A.

FIG 4

TVV2 capsid coordinates genome arrangement and egression of transcription intermediates. (A) 10 Å thick slab view of the Icosahedral viral capsid of arbitrary contour colored in grayscale with map densities in white and 0-values in black. Sites of icosahedral symmetry through the slab are labeled with thin, white arrows, and thick, white arrows indicate the location of the C terminus based on the atomic model. Distance between dsRNA strands measures ∼30 Å. (B) Cutaway view of capsid interior with electrostatic potential diagrams illustrating positive (blue) and negative (red) surfaces. I5 vertices are marked with arrows (blue). (C) I5 vertex as viewed from the capsid exterior; CSP-A K157, R159, and N255 residues are shown as ball and stick diagrams. (D) Exterior view of the TVV2 capsid protein decamer fit into the icosahedral map (radially colored) with surface colored by electrostatic potential (left). 90^° rotation of A and cutaway view of the 5-fold channel, with local residues from a CSP-A subunit (K157, R159, and N255) and channel diameters labeled (middle). Interior view of TVV2 decamer surface fit into map (right). (E) Exterior view of the dsRNA virus CPV CSP in the quiescent state (RCSB: 3JAZ) decamer fit into the icosahedral map with surface colored based on electrostatic potential (left). A cutaway view of the 5-fold channel, with involved residues from an CSP-A conformer (K674 and Q673) and channel diameters labeled (middle). Interior view of CPV CSP decamer fit into map (right).

FIG 5

Structural comparison of TVV2 CSP to ScV-L-A Gag. (A) Exterior view of ScV-L-A (yellow) and TVV2 (magenta) capsid shell proteins displayed with secondary structural elements of similar position labeled. (B) Orthogonal view of similar secondary structural elements between TVV2 and ScV-L-A capsid shell proteins superposed with outlines of the nonconserved segments. (C) Zoomed in view of ScV-L-A GTase site colored in yellow (left) with residues implicated in enzymatic activity (Y150, D152, K156, K158 Y452, D540, Y538, K536, R567, R568, and R570) colored green and the essential His154 labeled in orange (left). The labeled and putative GTase site of TVV2 is colored in magenta (right) with local residues colored green and labeled (R74, Y368, T369, K523, R525, H537, K539, R646, H648, R653, Y655, and H658) (right). (D) Exterior view of electrostatic potential maps of ScV-L-A (left) and TVV2 (right) CSP monomers, with negative (red) and positive (blue) charge distributions. Opacity changed to illustrate residue locations. (E) Enzymatic sites from panel C superposed. (F) Close-up views of the sites from panel C with surfaces colored based on electrostatic potential.