Structure of the herpes-simplex virus portal-vertex

Herpesviruses include many important human pathogens such as herpes simplex virus, cytomegalovirus, varicella-zoster virus, and the oncogenic Epstein–Barr virus and Kaposi sarcoma–associated herpesvirus. Herpes virions contain a large icosahedral capsid that has a portal at a unique 5-fold vertex, similar to that seen in the tailed bacteriophages. The portal is a molecular motor through which the viral genome enters the capsid during virion morphogenesis. The genome also exits the capsid through the portal-vertex when it is injected through the nuclear pore into the nucleus of a new host cell to initiate infection. Structural investigations of the herpesvirus portal-vertex have proven challenging, owing to the small size of the tail-like portal-vertex–associated tegument (PVAT) and the presence of the tegument layer that lays between the nucleocapsid and the viral envelope, obscuring the view of the portal-vertex. Here, we show the structure of the herpes simplex virus portal-vertex at subnanometer resolution, solved by electron cryomicroscopy (cryoEM) and single-particle 3D reconstruction. This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures that occupy the sites normally taken by the penton and the Ta triplex. Our data revealed that the PVAT is composed of 10 copies of the C-terminal domain of pUL25, which are uniquely arranged as two tiers of star-shaped density. Our 3D reconstruction of the portal-vertex also shows that one end of the viral genome extends outside the portal in the manner described for some bacteriophages but not previously seen in any eukaryote viruses. Finally, we show that the viral genome is consistently packed in a highly ordered left-handed spool to form concentric shells of DNA. Our data provide new insights into the structure of a molecular machine critical to the biology of an important class of human pathogens

In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging

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

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)

RONIN Is an Essential Transcriptional Regulator of Genes Required for Mitochondrial Function in the Developing Retina

SummaryA fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

DNA packaging in HSV-1.

<p>The unsharpened C5 reconstruction of HSV-1 is presented showing a series of radially cropped views of the interior density; these reveal the highly ordered arrangement of packaged DNA. The outermost (a), second (c), and third (e) shells are shown, revealing a left-handed spool of density. Spherical sections of each shell are also presented (b,d,f). HSV, Herpes Simplex Virus.</p

The structure of the portal-vertex interior.

<p>A central slice through the C5 reconstruction of the HSV-1 virion reveals the internal features of the portal-vertex (a). Notably, a strong linear density is seen to run through the portal-vertex that we attribute to genomic DNA (white arrow). The outermost feature, the PVAT, is weakly resolved as fuzzy density, suggesting that this feature is not well constrained. Isosurface representation of the unsharpened map presents a clearer representation of the PVAT (b), while in the sharpened density map, the packaged DNA is not seen, revealing the interior features of the capsid shell (c). A clipped, close-up view of the portal-vertex (boxed in c) highlights the morphology of the portal (pUL6) and, lying between the portal and the PVAT, the pentameric portal-vertex protein (wall-eyed stereo pair view, d). A close-up stereo view of the pentameric portal-vertex protein (boxed in d) clearly shows the density that is consistent with a two-helix coiled-coil motif (pink arrow, e). The density running through the centre of the portal-vertex that we attribute to DNA is also clearly visible (blue arrow). The density map was segmented to highlight three features: the portal (mauve), the pentameric portal-vertex protein (purple), and the periportal triplex–like density (magenta). The segmented portal-vertex is presented as stereo views both perpendicular to (e) and along (f) the portal axis. In panel e, the capsid and triplex-like assemblies are clipped to expose the pentameric portal-vertex protein; this and the portal are not clipped. In panel f, the pUL25/PVAT component is clipped away to expose the underlying features. HSV, Herpes Simplex Virus; PVAT, portal-vertex–associated tegument.</p

Stereo pair views of the structure and composition of the PVAT.

<p>Atomic coordinates for the CATC components pUL17, pUL25, and pUL36 (PDB 6CGR [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006191#pbio.2006191.ref005" target="_blank">5</a>]) and the C-terminal domain of pUL25 (PDB 2F5U [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006191#pbio.2006191.ref038" target="_blank">38</a>]) were docked into the portal-vertex density (a). Each pentonal five-helix CATC bundle has been shown to include two copies of an N-terminal α-helix of pUL25 (blue) along with two copies of a C-terminal α-helix of pUL36 (pink); these are bound to pUL17 (orange) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006191#pbio.2006191.ref005" target="_blank">5</a>]. The atomic model of the penton-vertex CATC matches well the equivalent density at the portal-vertex, indicating that there are likely a total of 10 copies of pUL25 at the portal-vertex as well. The PVAT assembly comprises 10 globular densities arranged as two C5 symmetric star-shaped rings that crown the portal-vertex. We docked five copies of the atomic model for the C-terminal domain of pUL25 into the proximal (inner) tier (blue, b). The docked coordinates were then saved as a single model that was docked into the less well-defined distal tier (c). A side view of the CATC/PVAT components is shown (d). CATC, capsid-associated tegument complex; PVAT, portal-vertex–associated tegument.</p