RF3-induced subunit rotation destabilizes RF1 binding. a Cryo-EM map of SSU (light blue) and RF1 (orange) from state III compared with SSU (dark blue) and RF1 (red) from state IV. b Isolated cryo-EM electron densities (grey mesh) with molecular models for RF1 from state III (orange) and state IV (red) shown at the same contour level based on comparison with the SSU density. c Domain 2/4 of RF1 from state III (sIII:RF1, orange) is rotated by 6° and shifted by 4 Å compared to RF1 from state IV (sIV:RF1, red). d, e Contacts (arrowed) between RF1 (orange) and P/P-tRNA (green) are lost upon formation of the hybrid P/E-tRNA (light blue). Amino acids of RF1 that contact P/P-tRNA are shown as spheres. e Zoom of d showing the presence or absence of RF1 contacts with the ASL of P/P- or P/E-tRNA, respectively. 

Daniel N. Wilson Research Group

Significance

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome. 

Graf, M., Huter, P., Maracci, C.,  Peterek, M., Rodnina, M.V.,  and Wilson D.N.: Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1 Nat. Commun. (2018)9, 3053 doi:10.1038/s41467-018-05465-1

Structure of the MiCP and its interaction with other capsid proteins. (A) Structure of the MiCP shown in stick representation with carbon atoms in magenta. The first and last structured residues of MiCP are labeled. The electron density map of the MiCP contoured at 2σ is shown as a blue mesh. Major capsid proteins are shown in cartoon representation with VP1 in blue, VP2 in green, and VP3 in red. (B and C) Comparison of capsid proteins VP2 of SBV (B) and poliovirus 1 (C). The MiCP of SBV, which is highlighted in magenta (B), occupies the volume of the puff loop in VP2 of poliovirus 1, highlighted in orange (C). (D and E) The VP3 subunit of SBV (D) lacks the knob loop present in poliovirus 1 (E), highlighted in green

Pavel Plevka Research group

Significance

Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 Å and 12 Å in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm.

Procházková, M. et al.: Virion structure and genome delivery mechanism of sacbrood honeybee virus. PNAS 2018, 115 (30) 7759-7764. doi.org/10.1073/pnas.1722018115.