Calicivirus VP2 forms a portal-like assembly following receptor engagement

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly—which was not detected in undecorated virions—is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae

Microwave hyperthermia represses human papillomavirus oncoprotein activity and induces cell death due to cell stress in 3D tissue models of anogenital precancers and cancers

Background: Hyperthermia is a well-accepted cancer therapy. Microwaves provide a very precise, targeted means of hyperthermia and are currently used to treat plantar warts caused by cutaneous-infective human papillomaviruses (HPVs). Other HPV genotypes infecting the anogenital mucosa cause genital warts or preneoplastic lesions or cervical cancer. Effective, non-ablative therapies for these morbid HPV-associated lesions are lacking. Methods: The molecular consequences of microwave treatment were investigated in in vitro cultured three-dimensional HPV-positive cervical tumour tissues, and tissues formed from HPV-infected normal immortalised keratinocytes. Microwave energy delivery to tissues was quantified. Quantitative reverse transcriptase PCR was used to quantify mRNA expression. Immunohistochemistry and fluorescence immunostaining was used to assess protein expression. Findings: Microwave energy deposition induced sustained, localised cell death at the treatment site. There was a downregulation in levels of HPV oncoproteins E6 and E7 alongside a reduction in cellular growth/proliferation and induction of apoptosis/autophagy. HSP70 expression confirmed hyperthermia, concomitant with induction of translational stress. Interpretation: The data suggest that microwave treatment inhibits tumour cell proliferation and allows the natural apoptosis of HPV-infected cells to resume. Precision microwave delivery presents a potential new treatment for treating HPV-positive anogenital precancerous lesions and cancers. Funding: Funding was through an Innovate UK Biomedical Catalyst grant (ID# 92138-556187), a Chief Scientist Office grant (TCS/19/11) and core support from Medical Research Council (MC_ UU_12014) core funding for the MRC-University of Glasgow Centre for Virus Research

Calicivirus VP2 forms a portal-like assembly following receptor engagement.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.IG is a Wellcome Senior Fellow (Ref: 207498/Z/17/Z). M.J.C. was supported by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC WestBIO DTP: BB/J013854/1). D.B and M.M. are supported by the UK Medical Research Council (MC_UU_12014/7)

Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus

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

The Cryo-EM Structure of Vesivirus 2117 Highlights Functional Variations in Entry Pathways for Viruses in Different Clades of the Vesivirus Genus

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 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’, which plays an important role in both 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