Capsid Structure of Leishmania RNA Virus 1

Abstract

Leishmania parasites cause a variety of symptoms, including mucocutaneous leishmaniasis, which results in the destruction of the mucous membranes of the nose, mouth, and throat. The species of Leishmania carrying Leishmania RNA virus 1 (LRV1), from the family Totiviridae, are more likely to cause severe disease and are less sensitive to treatment than those that do not contain the virus. Although the importance of LRV1 for the severity of leishmaniasis was discovered a long time ago, the structure of the virus remained unknown. Here, we present a cryo-electron microscopy reconstruction of the virus-like particle of LRV1 determined to a resolution of 3.65 Å. The capsid has icosahedral symmetry and is formed by 120 copies of a capsid protein assembled in asymmetric dimers. RNA genomes of viruses from the family Totiviridae are synthetized, but not capped at the 5' end, by virus RNA polymerases. To protect viral RNAs from degradation, capsid proteins of the L-A totivirus cleave the 5' caps of host mRNAs, creating decoys to overload the cellular RNA quality control system. Capsid proteins of LRV1 form positively charged clefts, which may be the cleavage sites for the 5' cap of Leishmania mRNAs. The putative RNA binding site of LRV1 is distinct from that of the related L-A virus. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative decapping site. Such inhibitors may be developed into a treatment for mucocutaneous leishmaniasis caused by LRV1-positive species of LeishmaniaIMPORTANCE Twelve million people worldwide suffer from leishmaniasis, resulting in more than 30 thousand deaths annually. The disease has several variants that differ in their symptoms. The mucocutaneous form, which leads to disintegration of the nasal septum, lips, and palate, is caused predominantly by Leishmania parasites carrying Leishmania RNA virus 1 (LRV1). Here, we present the structure of the LRV1 capsid determined using cryo-electron microscopy. Capsid proteins of a related totivirus, L-A virus, protect viral RNAs from degradation by cleaving the 5' caps of host mRNAs. Capsid proteins of LRV1 may have the same function. We show that the LRV1 capsid contains positively charged clefts that may be sites for the cleavage of mRNAs of Leishmania cells. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative mRNA cleavage site. Such inhibitors may be used as treatments for mucocutaneous leishmaniasis.

Keywords: CAP-4; LRV1; Leishmania; RNA; Totiviridae; Viannia; capsid; cryo-electron microscopy; decapping; genome; leishmaniasis; mRNA; parasite; structure; uncoating; virion; virus.

FIG 1

Structure of the LRV1 capsid. (A) Surface representation of cryo-EM reconstruction of the LRV1 capsid, rainbow colored based on distance from particle center. The front bottom right octant of the particle has been removed to show the interior. Clipped density is shown in gray. (B) Organization of the LRV1 capsid, with subunit A shown in red and subunit B shown in green. The borders of one icosahedral asymmetric unit are outlined with a black triangle, with the positions of the 5-fold axis indicated as a pentamer, the 3-fold axes as triangles, and the 2-fold axis as an oval. Scale bar, 10 nm.

FIG 2

Structure of LRV1 capsid proteins. (A) Cartoon representation of two capsid proteins of LRV1 forming icosahedral asymmetric unit. Subunit A is shown in red, and subunit B in shown in green. Divisions of the subunits into alpha and beta domains are indicated by blue dashed lines. The positions of icosahedral symmetry axes are indicated by an oval for 2-fold, a triangle for 3-fold, and a pentagon for 5-fold. Residues 15 to 18, 291 to 299, 518 to 520, and 577 to 582, which are resolved in subunit A but not in B, are highlighted in cyan. Residues 205 to 209 and 636 to 642, which are resolved in subunit B but not in subunit A, are highlighted in magenta. (B) Scheme showing parts of subunits A (red) and B (green) that are resolved in cryo-EM reconstruction of the LRV1 capsid. Parts of the proteins that were not resolved are shown in gray. (C) Diagram of secondary structure elements of the LRV1 capsid protein. α-Helices are shown as cylinders numbered from 1 to 15, and β-strands are shown as arrows labeled A to L. β-Strands J and K, which are resolved in subunit A but not in subunit B of LRV1, are shown in blue. Secondary structure elements that are shared between LRV1 and L-A virus are highlighted in red.

FIG 3

Intersubunit interfaces in the LRV1 capsid. (A) Diagram of interaction interfaces in the LRV1 capsid. Subunits A are shown in red, and subunits B are shown in green. The subunits are numbered to indicate to which icosahedral asymmetric unit they belong. (B) Table of buried surface areas of intersubunit interfaces as indicated in panel A of LRV1 and L-A virus.

FIG 4

Pores in the LRV1 capsid. (A to C) Cartoon representations of capsid proteins of LRV1 forming pores on 5-fold (A) and 3-fold (B) symmetry axes. An additional pore is formed at the intersubunit interface approximately in the middle between the 3-fold and 2-fold symmetry axes of the capsid (C). Subunits A are shown in red, and subunits B are shown in green. Side chains of residues forming the narrowest constriction in the pore are shown in stick representation. (D to F) Surface representation of pores colored according to charge distribution. Blue indicates positive charge, white is neutral, and red is negative.

FIG 5

Comparison of capsids and inner capsid shells of viruses that replicate their double-stranded RNA genomes inside particles. (A to E) Surface representations of LRV1 (A), L-A virus (B) (18), phage phi6 (C) (44), inner core of rotavirus A (D) (79), and inner core of reovirus (E) (80). Subunits A are shown in red, and subunits B are shown in green. Scale bar, 10 nm. (F to J) Cartoon representation of proteins forming icosahedral asymmetric units of viruses. Parts of capsid proteins of L-A virus that can be superimposed onto the LRV1 structure are highlighted in cyan and magenta in subunits A and B, respectively (G). Positions of icosahedral symmetry axes are indicated by pentamers for 5-fold, triangles for 3-fold, and ovals for 2-fold axis.

FIG 6

Putative mRNA cap cleavage site of LRV1 is distinct from that of L-A virus. (A and B) Molecular surfaces of icosahedral asymmetric units colored according to charge distribution of LRV1 (A) and L-A virus (B). (A) Putative mRNA binding sites of LRV1 are indicated by black rectangles, and positions corresponding to the decapping sites of L-A virus are indicated by dotted rectangles. (B) Positions of RNA decapping sites in L-A virus are indicated by dashed rectangles. (C and D) Comparison of structures of putative cap cleavage site in L-A virus (D) and the corresponding region in LRV1 (C). (E and F) Details of putative RNA decapping sites in subunits A (E) and B (F) of LRV1. Subunits A are shown in red, and subunits B are shown in green.

FIG 7

Identification of putative 7-methyl-guanosine cap binding site in the LRV1 capsid. (A) Molecular surface representation of part of the LRV1 capsid. Subunits A are shown in red, and subunits B are shown in green. Cyan spheres indicate positions of the central phosphate in m7Gppp of CAP-4 molecules docked to the structure. The docked molecules cluster in the positively charged clefts of LRV1 capsid proteins and in the pores of the capsid. (B and C) Examples of results from docking of CAP-4 structure that position the m7Gppp of CAP-4 in a position exposed to nucleophilic attack by the amide nitrogen atom from the side chain of His410 (B) or His59 (C).