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Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry. Author summary: To infect a cell, non-enveloped viruses must have a mechanism to transfer their genomes across a membrane. The double-strand RNA (dsRNA) viruses deliver into the cytosol not just their genomes but an intact subviral particle (diameter ~70 nm). Rhesus rotavirus (RRV) particles attach through a spike-like outer-layer protein, VP4 (cleaved to VP5* and VP8*), then wrap themselves in membrane at the cell surface, producing a small, cytosolic vesicle surrounding each virion, from which the subviral particles escape directly. Loss of Ca2+ from the entering virion always precedes escape. In previous work we found that a conformational change in VP5*/VP8* projects the C-terminal "foot" domain of VP5* outward. We now show, by combining cryo-EM and single-particle optical microscopy of RRV particles interacting with liposomes, that the outward-projected foot penetrates the lipid bilayer, permeabilizing it to Ca2+. The new experiments thus recapitulate the initial step in particle escape and show that the conformational change must occur while the spike protein is membrane-attached, to couple the VP5* conformational change with membrane insertion. These findings bring us closer to a full molecular description how a large, subviral particle (about 70 nm diameter) can be delivered into a target host cell. [ABSTRACT FROM AUTHOR]

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines. [ABSTRACT FROM AUTHOR]

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry. Author summary: To infect a cell, non-enveloped viruses must have a mechanism to transfer their genomes across a membrane. The double-strand RNA (dsRNA) viruses deliver into the cytosol not just their genomes but an intact subviral particle (diameter ~70 nm). Rhesus rotavirus (RRV) particles attach through a spike-like outer-layer protein, VP4 (cleaved to VP5* and VP8*), then wrap themselves in membrane at the cell surface, producing a small, cytosolic vesicle surrounding each virion, from which the subviral particles escape directly. Loss of Ca2+ from the entering virion always precedes escape. In previous work we found that a conformational change in VP5*/VP8* projects the C-terminal "foot" domain of VP5* outward. We now show, by combining cryo-EM and single-particle optical microscopy of RRV particles interacting with liposomes, that the outward-projected foot penetrates the lipid bilayer, permeabilizing it to Ca2+. The new experiments thus recapitulate the initial step in particle escape and show that the conformational change must occur while the spike protein is membrane-attached, to couple the VP5* conformational change with membrane insertion. These findings bring us closer to a full molecular description how a large, subviral particle (about 70 nm diameter) can be delivered into a target host cell. [ABSTRACT FROM AUTHOR]

Flavivirus envelope (E) and precursor M (prM) proteins, when ectopically expressed, assemble into empty, virus-like particles (VLPs). Cleavage of prM to M and loss of the pr fragment converts the VLPs from immature to mature particles, mimicking a similar maturation of authentic virions. Most of the VLPs obtained by prM-E expression are smaller than virions; early, low-resolution cryo-EM studies suggested a simple, 60-subunit, icosahedral organization. We describe here the cryo-EM structure of immature, small VLPs (smVLPs) from dengue virus type 2 and show that they have octahedral rather than icosahedral symmetry. The asymmetric unit of the octahedral particle is an asymmetric trimer of prM-E heterodimers, just as it is on icosahedral immature virions; the full, octahedrally symmetric particle thus has 24 such asymmetric trimers or 72 prM-E heterodimers in all. Cleavage of prM and release of pr generates ovoid, somewhat irregular, mature particles. Previous work has shown that mature smVLPs have fusion properties identical to those of virions, consistent with local, virion-like clustering of 36 E dimers on their surface. The cryo-EM structure and the properties of the smVLPs described here relate directly to ongoing efforts to use them as vaccine immunogens., Importance: Ectopic expression of flavivirus envelope (E) and precursor M (prM) proteins leads to the formation and secretion of empty, virus-like particles (VLPs). We show that a major class of VLPs, of smaller diameter than those of virion size ("small VLPs": smVLPs), are octahedrally symmetric particles. The known characteristics of immature virions (asymmetric trimers of prM-E heterodimers) allow us to understand the assembly of an octahedral (rather than icosahedral) surface lattice. Cleavage of prM and formation of mature, fusogenic smVLPs yield somewhat irregular, ovoid particles. These observations are directly relevant to proposals for using immunogenic but non-infectious VLPs as components of specific flavivirus vaccines.

Cells coordinate diverse events at anaphase onset, including separase activation, cohesin cleavage, chromosome separation, and spindle reorganization. Regulation of the XMAP215 family member and microtubule polymerase, Stu2, at the metaphase-anaphase transition determines a specific redistribution from kinetochores to spindle microtubules. We show that cells modulate Stu2 kinetochore-microtubule localization by Polo-like kinase1/Cdc5-mediated phosphorylation of T866, near the Stu2 C-terminus, thereby promoting dissociation from the kinetochore Ndc80 complex. Cdk/Cdc28 likely primes Cdc5:Stu2 interaction. Cdc28 activity is also required for Stu2 nuclear import. PP2A Cdc55 actively opposes Cdc5 activity on Stu2 T866 during metaphase. This counter-regulation allows for switchlike redistribution of Stu2 pT866 at anaphase onset when separase inhibits PP2A Cdc55 . Blocking Stu2 T866 phosphorylation disrupts anaphase spindle progression, and we infer that PP2A Cdc55 regulates the mitotic spindle by dephosphorylating Stu2 and other MAPs. These data support a model in which increased phosphorylation at anaphase onset results from phosphatase inhibition and point to a larger regulatory network that facilitates rapid cytoskeletal modulation required for anaphase spindle maintenance.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca 2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca 2+ -stabilized trimer; loss of Ca 2+ releases both outer-layer proteins from the particle. The other outer-layer protein, VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca 2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca 2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.