Search

Interactions between respiratory viruses during infection affect transmission dynamics and clinical outcomes. To identify and characterize virus-virus interactions at the cellular level, we coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy, live-cell imaging, scanning electron microscopy and cryo-electron tomography revealed extracellular and membrane-associated filamentous structures consistent with hybrid viral particles (HVPs). We found that HVPs harbour surface glycoproteins and ribonucleoproteins of IAV and RSV. HVPs use the RSV fusion glycoprotein to evade anti-IAV neutralizing antibodies and infect and spread among cells lacking IAV receptors. Finally, we show that IAV and RSV coinfection in primary cells of the bronchial epithelium results in viral proteins from both viruses co-localizing at the apical cell surface. Our observations define a previously unknown interaction between respiratory viruses that might affect virus pathogenesis by expanding virus tropism and enabling immune evasion.

Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are thought to provide a favourable environment for replication to occur, concentrating essential viral structural and nonstructural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3Dpol), we report fibril structures, solved at ~7-9 Å resolution by cryo-EM, revealing multiple conformations of a flexible assembly. Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher-order 3Dpol complexes as a dynamic scaffold within which RNA replication can occur., Loundras et al. report on the fibril components of the RNA-dependent RNA polymerase RdRp from foot-and-mouth disease virus. They demonstrate that higher-order fibril-based interactions create multiple complex structures within which RNA replication can occur.

Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (I

The Fanconi Anaemia pathway operates for the repair of interstrand crosslinks and the maintenance of genomic stability upon replication stalling. Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step in this pathway. Evidence suggests that FANCD2 ubiquitination precedes FANCI ubiquitination, and that both the FANCD2-ubiquitinated (ID2Ub) and the di- monoubiquitinated (IUbD2Ub) complex clamp on DNA. However, FANCD2 is deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUbD2). Here, we present a 4.1 Å cryo-EM structure of IUbD2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. While the target lysine of FANCD2 (K561) is partially buried in the non-ubiquitinated ID2-DNA complex, it becomes fully exposed in the IUbD2-DNA structure, and thus can be ubiquitinated at a faster rate. The IUbD2-DNA complex cannot easily revert to the non-ubiquitinated ID2 state, due to USP1-UAF1-resistance, conferred by the presence of DNA and FANCD2. ID2Ub-DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. FANCI ubiquitination also progresses at a faster rate in ID2Ub-DNA over ID2-DNA complex, and results in partial DNA-dependent protection from FANCD2 deubiquitination. Taken together, our results suggest that, while FANCD2 ubiquitination promotes FANCI ubiquitination, FANCI ubiquitination in turn maintains FANCD2 ubiquitination by two mechanisms: it prevents excessive FANCD2 deubiquitination within an IUbD2Ub-DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUbD2 complex.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.

Interactions between respiratory viruses impact viral transmission dynamics and clinical outcomes. To identify and characterize virus-virus interactions at the cellular level, we coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy, live-cell imaging, scanning electron microscopy, and cryo-electron tomography revealed extracellular and membrane-associated filamentous structures consistent with hybrid viral particles (HVPs). We show that HVPs harbor surface glycoproteins and ribonucleoproteins of IAV and RSV, and use the RSV fusion glycoprotein to evade anti-IAV neutralising antibodies and to infect and spread among cells lacking IAV receptors. Finally, we show evidence of IAV and RSV coinfection within cells of the bronchial epithelium, with viral proteins from both viruses co-localising at the apical surface. Our observations have profound implications for infection biology as they define a previously unknown interaction between respiratory viruses that might affect virus pathogenesis by expanding virus tropism and facilitating immune evasion.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Treatments for RSV disease are however limited and efforts to produce an effective vaccine have so far been unsuccessful. Understanding RSV virion structure is an important prerequisite for developing interventions to treat or prevent infection but has been challenging because of the fragility of virions propagated in cell culture. Here we show, using cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) of RSV particles cultivated directly on transmission electron microscopy (TEM) grids, that there is extensive helical symmetry in RSV filamentous virions. We have calculated a 16 Å resolution three-dimensional reconstruction of the viral envelope, targeting the matrix protein (M) that forms an endoskeleton below the viral membrane. These data define a helical lattice of M proteins, showing how M is oriented relative to the viral envelope and that helical ordering of viral glycoproteins that stud the viral envelope is coordinated by the M layer. Moreover, the helically ordered viral glycoproteins in RSV filamentous virions cluster in pairs, which may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV filamentous virions. Overall, the structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly.

Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine virus (VESV) clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense-RNA-containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22-amino-acid insertion in the sequence of the FCV major capsid protein that forms a “cantilevered arm” that both plays an important role in receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the Vesivirus genus. IMPORTANCE Vesivirus 2117 has caused significant losses in manufacturing of biopharmaceutical products following contamination of cell cultures used in their production. We report the structure of the vesivirus 2117 capsid, the shell that encloses the virus’s genome. Comparison of this structure with that of a related vesivirus, feline calicivirus (FCV), highlighted potentially important differences related to virus attachment and entry. Our findings suggest that these two viruses may bind differently to receptors at the host cell surface. We also show that a region of the capsid protein of FCV that rearranges following receptor engagement is not present in vesivirus 2117. These structural changes in the FCV capsid have been shown to allow the assembly of a portal-like structure that is hypothesized to deliver the viral genome to the cell’s interior. Our data suggest that the 2117 portal assembly may employ a different means of anchoring to the outer face of the capsid.

Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are believed to provide a favourable environment for replication to occur, concentrating essential viral structural and non-structural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3Dpol), we report fibril structures, solved at ~7-9 Å resolution by cryo-EM, revealing multiple conformations of a flexible assembly. Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher order 3Dpol complexes as a dynamic scaffold within which RNA replication can occur.

The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double‐stranded DNA via promoting a large‐scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary “Arm” ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1‐UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post‐translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA., The FANCI‐FANCD2 (ID2) complex is involved in DNA repair. The ubiquitination of each protein acts to maintain the complex on double stranded DNA, but through different mechanisms.

Weibel-Palade bodies (WPBs) are secretory granules that contain von Willebrand factor and P-selectin, molecules that regulate hemostasis and inflammation, respectively. The presence of CD63/LAMP3 in the limiting membrane of WPBs has led to their classification as lysosome-related organelles. Many lysosome-related organelles contain intraluminal vesicles (ILVs) enriched in CD63 that are secreted into the extracellular environment during cell activation to mediate intercellular communication. To date, there are no reports that WPBs contain or release ILVs. By light microscopy and live-cell imaging, we show that CD63 is enriched in microdomains within WPBs. Extracellular antibody recycling studies showed that CD63 in WPB microdomains can originate from the plasma membrane. By cryo-electron tomography of frozen-hydrated endothelial cells, we identify internal vesicles as novel structural features of the WPB lumen. By live-cell fluorescence microscopy, we directly observe the exocytotic release of EGFP-CD63 ILVs as discrete particles from individual WPBs. WPB exocytosis provides a novel route for release of ILVs during endothelial cell stimulation.

Filopodia are cellular projections around 100nm in diameter and usually several micrometres in length. They are composed of fascin-bundled actin filaments and the molecular motor myosin‑10 localises at the extreme tip. Filopodia are important in cell migration and in making cell-cell contacts and have been linked with roles in diverse areas of biology including angiogenesis, and path-finding properties of neuronal growth cones. Filopodia can be induced in model cell lines by overexpresion of myosin‑10 and live-cell imaging shows that they undergo stochastic cycles of extension and retraction. Myosin‑10 transports cargo along the actin filament bundle towards the filopodial tip, forming a complex of proteins which are responsible for governing growth, retraction and adhesion. We have used a combination of optical and electron microscopy to study the dynamics and ultra-structure of myosin‑10 induced filopodia. Electron cryo-tomography of flash-frozen vitrified mammalian cells revealed the arrangement of actin filaments within the filopodia. Single molecule imaging by total internal reflection fluorescence microscopy allowed movement of myosin‑10 with the filopodia to be quantified and super-resolution localisation gave structural and mechanical information about the filopodium that could be correlated to our electron tomographic images.