Structural Insights into Lactococcal Siphophage p2 Baseplate Activation Mechanism

Virulent phages infecting L. lactis, an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca2+. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.

Bovine Papillomatosis Hiding a Zoonotic Infection: Epitheliotropic Viruses in Bovine Skin Lesions

We describe two cases of skin co-infections with epitheliotropic viruses, detected in two cattle during lumpy skin disease (LSD) surveillance in northern Italy. A diagnostic protocol including different molecular methods as well as negative staining electron microscopy was applied to detect the most common viral agents belonging to the family Papillomaviridae, Poxviridae and Herpesviridae which cause skin diseases in cattle. Two specimens were collected from cases clinically diagnosed as papillomatosis and pseudo-LSD. Both skin lesions were shown to harbor more than one viral species. This case report shows, for the first time, co-infection of zoonotic parapoxvirus with bovine papillomavirus and herpesvirus in skin lesions of cattle. In particular, the simultaneous presence of virions morphologically referable to parapoxvirus and papillomavirus confirms that the replication of both viruses in the same lesion can happen and the so-called papillomatosis can bear zoonotic viruses.

Bovine Papillomavirus 1 Gets Out of the Flock: Detection in an Ovine Wart in Sicily

A proliferative cauliflower lesion was excised from the udder of a sheep. Histological investigation confirmed the macroscopic classification of the lesion as a papilloma, without any fibroblastic proliferation. PCR revealed the presence of bovine papillomavirus (BPV), which was further confirmed by the identification of a Deltapapillomavirus 4 by Next Generation Sequencing analysis. This was subsequently classified as bovine papillomavirus type 1. Negative staining electron microscopy (EM) analyses produced negative test results for papillomavirus particles. RNA in situ hybridization (ISH) confirmed the presence of BPV-1. The results further confirm the ability of BPVs belonging to the Deltapapillomavirus genus to infect distantly related species and to cause lesions that are different from sarcoids.

Filamentous versus Spherical Morphology: A Case Study of the Recombinant A/WSN/33 (H1N1) Virus

Influenza A virus is a serious human pathogen that assembles enveloped virions on the plasma membrane of the host cell. The pleiomorphic morphology of influenza A virus, represented by spherical, elongated, or filamentous particles, is important for the spread of the virus in nature. Using fixative protocols for sample preparation and negative staining electron microscopy, we found that the recombinant A/WSN/33 (H1N1) (rWSN) virus, a strain considered to be strictly spherical, may produce filamentous particles when amplified in the allantoic cavity of chicken embryos. In contrast, the laboratory WSN strain and the rWSN virus amplified in Madin–Darby canine kidney cells exhibited a spherical morphology. Next-generation sequencing (NGS) suggested a rare Ser126Cys substitution in the M1 protein of rWSN, which was confirmed by the mass spectrometric analysis. No structurally relevant substitutions were found by NGS in other proteins of rWSN. Bioinformatics algorithms predicted a neutral structural effect of the Ser126Cys mutation. The mrWSN_M1_126S virus generated after the introduction of the reverse Cys126Ser substitution exhibited a similar host-dependent partially filamentous phenotype. We hypothesize that a shortage of some as-yet-undefined cellular components involved in virion budding and membrane scission may result in the appearance of filamentous particles in the case of usually “nonfilamentous” virus strains.

Structural study of Topoisomerase IV-DNA-Levofloxacin complexes from Streptococcus pneumoniae

S. pneumoniae is an important pathogen causing pulmonary infection, acute otitis media and purulent meningitis in infants and children. Type II topoisomerases are enzymes that play essential roles in DNA replication, chromosome segregation and recombination throughout all living organisms. Topoisomerases IV can make a transient break in DNA strands in one chromosome. These enzymes are very important antibacterial as well as anticancer targets and potential anti-trypanosomal targets. Levofloxacin has shown efficient inhibition of Type II topoisomerases in S. pneumoniae. Its mechanism of action is to inhibit the activity of DNA topoisomerase, prevent bacterial DNA synthesis and replication leading to bacterial death. We focused on solving the key structures of topoisomerase IV-DNA-levofloxacin complexes by negative staining electron microscopy and the resulting model was obtained at 32 Å by 3D autorefine in Relion 3.0. This study was to try and obtain the structure of the whole complex with DNA bound in the G-gate and the T-gate in order to study DNA capture and transport in type II topoisomerases.

Domain topology of human Rasal

Rasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BPand Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BPand Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

A closed conformation of the Caenorhabditis elegans separase–securin complex

The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.