Cryo-electron Microscopy Study of the Genome Release of the Dicistrovirus Israeli Acute Bee Paralysis Virus

Abstract

Viruses of the family Dicistroviridae can cause substantial economic damage by infecting agriculturally important insects. Israeli acute bee paralysis virus (IAPV) causes honeybee colony collapse disorder in the United States. High-resolution molecular details of the genome delivery mechanism of dicistroviruses are unknown. Here we present a cryo-electron microscopy analysis of IAPV virions induced to release their genomes in vitro We determined structures of full IAPV virions primed to release their genomes to a resolution of 3.3 Å and of empty capsids to a resolution of 3.9 Å. We show that IAPV does not form expanded A particles before genome release as in the case of related enteroviruses of the family Picornaviridae The structural changes observed in the empty IAPV particles include detachment of the VP4 minor capsid proteins from the inner face of the capsid and partial loss of the structure of the N-terminal arms of the VP2 capsid proteins. Unlike the case for many picornaviruses, the empty particles of IAPV are not expanded relative to the native virions and do not contain pores in their capsids that might serve as channels for genome release. Therefore, rearrangement of a unique region of the capsid is probably required for IAPV genome release.

Importance: Honeybee populations in Europe and North America are declining due to pressure from pathogens, including viruses. Israeli acute bee paralysis virus (IAPV), a member of the family Dicistroviridae, causes honeybee colony collapse disorder in the United States. The delivery of virus genomes into host cells is necessary for the initiation of infection. Here we present a structural cryo-electron microscopy analysis of IAPV particles induced to release their genomes. We show that genome release is not preceded by an expansion of IAPV virions as in the case of related picornaviruses that infect vertebrates. Furthermore, minor capsid proteins detach from the capsid upon genome release. The genome leaves behind empty particles that have compact protein shells.

Keywords: Aparavirus; Apis mellifera; CCD; Dicistroviridae; Picornavirales; capsid; colony collapse disorder; cryo; electron microscopy; empty; genome; honey bee; honeybee; release; structure; uncoating; virion; virus.

FIG 1

Increased temperature triggers genome release of IAPV. (A) Thermal stability of IAPV virions. IAPV virions were mixed with Sybr green II dye and heated to the indicated temperatures (x axis). The fluorescence signal increased as the dye bound to RNA. Error bars indicate standard deviations of the means (n = 3). See Materials and Methods for details. Cryo-electron micrographs show native IAPV virions at room temperature (B) and empty IAPV particles (C), induced by incubating the virus at 63°C for 10 min. The black arrows in panel B indicate “lip-shaped” particles formed by IAPV capsid proteins. The white arrows indicate complexes of the honeybee protein hexamerin that contaminated the virus purification. Bars, 50 nm.

FIG 2

Structures of native IAPV virions and full and empty particles heated to 63°C. An X-ray structure of a native virion of IAPV (A) and cryo-EM structures of full virions heated to 63°C (B) and of empty particles prepared by heating virions to 63°C (C) are shown. The solvent-accessible surfaces are rainbow colored based on their distances from the particle center. Central slices of electron density maps of native virions (D), full virions heated to 63°C (E), and empty particles heated to 63°C (F) are shown in the bottom row. White areas indicate areas with high electron density values. The positions of selected icosahedral symmetry axes are labeled. The insets in panels D to F show details of electron density distributions near the 5-fold axes. The white arrow in the inset of panel F indicates a position of high electron density formed by putative ions. A corresponding density is not present in the virion structure, as indicated by a black arrow in the inset of panel E. Bars, 50 Å.

FIG 3

Comparison of icosahedral asymmetric units of IAPV virions and empty particles. Icosahedral asymmetric units of an IAPV virion heated to 63°C (A) and an empty particle (B) are shown as viewed from the particle center. VP1, VP2, VP3, and VP4 are shown in blue, green, red, and orange, respectively. The positions of 5-fold, 3-fold, and 2-fold icosahedral symmetry axes are indicated with pentagons, triangles, and ovals, respectively. The insets at bottom show representative electron densities of the IAPV virion and empty particle at resolutions of 3.26 Å and 3.85 Å, respectively. The maps are contoured at 4 σ.

FIG 4

Buried surface areas of interfaces within IAPV virions at different temperatures, within empty particles, and within pentamers. (A) Buried surface areas. Individual subunits are labeled according to their relative positions as shown in panel B. (B) Capsid surface representation of an IAPV virion, with VP1, VP2, and VP3 subunits shown in blue, green, and red, respectively. Icosahedral asymmetric units considered for buried surface calculations are labeled with letters. The buried surface areas were calculated using the PISA server (61). *, note that the differences in buried surface areas between the native and heated virions are due to the extra residues and side chains of VP4 that could be built into the heated virion structure. The differences do not correspond to major conformational changes.

FIG 5

Comparison of intersubunit interactions and charge distributions in IAPV virions, empty particles, and pentamers. Cartoon representations of an IAPV virion heated to 63°C (A), an empty particle (B), and a pentamer (C) are shown as viewed from the particle center. VP1 subunits are shown in blue, VP2 in green, VP3 in red, and VP4 in orange. Selected subunits are shown in bright colors. The positions of 5-fold, 3-fold, and 2-fold icosahedral symmetry axes are indicated with pentagons, triangles, and ovals, respectively. (D to F) Molecular surface of the capsid interior, colored according to the charge distribution. (G to I) Distributions of electron density close to the 5-fold axis. The maps are contoured at 4 σ.