A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions

Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture

Viral MicroRNA Effects on Pathogenesis of Polyomavirus SV40 Infections in Syrian Golden Hamsters

Shaojie Zhang, Vojtech Sroller, Preeti Zanwar, Steven J. Halvorson, Nadim J. Ajami, Corey W. Hecksel, Jody L. Swain, Connie Wong, Janet S. Butel, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of AmericaChun Jung Chen, Christopher S. Sullivan, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of AmericaJody L. Swain, Center for Comparative Medicine, Baylor College of Medicine, Houston, Texas, United States of AmericaEffects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.This work was supported in part by research grants R01 CA134524 (JSB) and R01 AI077746 (CSS) from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Molecular BiosciencesEmail: [email protected]

SuRVoS: Super-Region Volume Segmentation workbench

Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets

Machining protein microcrystals for structure determination by electron diffraction

We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells

Electron Bio-Imaging Centre (eBIC): the UK national research facility for biological electron microscopy.

The recent resolution revolution in cryo-EM has led to a massive increase in demand for both time on high-end cryo-electron microscopes and access to cryo-electron microscopy expertise. In anticipation of this demand, eBIC was set up at Diamond Light Source in collaboration with Birkbeck College London and the University of Oxford, and funded by the Wellcome Trust, the UK Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) to provide access to high-end equipment through peer review. eBIC is currently in its start-up phase and began by offering time on a single FEI Titan Krios microscope equipped with the latest generation of direct electron detectors from two manufacturers. Here, the current status and modes of access for potential users of eBIC are outlined. In the first year of operation, 222 d of microscope time were delivered to external research groups, with 95 visits in total, of which 53 were from unique groups. The data collected have generated multiple high- to intermediate-resolution structures (2.8-8 Å), ten of which have been published. A second Krios microscope is now in operation, with two more due to come online in 2017. In the next phase of growth of eBIC, in addition to more microscope time, new data-collection strategies and sample-preparation techniques will be made available to external user groups. Finally, all raw data are archived, and a metadata catalogue and automated pipelines for data analysis are being developed

Nuclear EGFR in breast cancer suppresses NK cell recruitment and cytotoxicity

Natural Killer (NK) cells can target and destroy cancer cells, yet tumor microenvironments typically suppress NK cell recruitment and cytotoxicity. The epidermal growth factor receptor (EGFR) is a potent oncogene that can activate survival, migration, and proliferation pathways, and clinical data suggests it may also play an immunomodulating role in cancers. Recent work has demonstrated a novel role for nuclear EGFR (nEGFR) in regulating transcriptional events unique from the kinase domain. Using a novel peptide therapeutic (cSNX1.3) that inhibits retrograde trafficking of EGFR and an EGFR nuclear localization mutant, we discovered that nEGFR suppresses NK cell recruitment and cytotoxicity. RNA-Seq analysis of breast cancer cells treated with cSNX1.3 or modified to lack a nuclear localization sequence (EGF

Cutting Edge: Building bridges between cellular and molecular structural biology

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures

The effects of scan patterns on secondary neutron dose equivalent in a uniform scanning proton therapy beam delivery system

Various scanning patterns of both circular and square shapes of proton beams have been tested to determine the resulting secondary neutron dose equivalent to a point representative of a point in a patient. The secondary neutron dose equivalent was measured while varying the scanning patterns\u27 size and the patient collimator size in two separate experiments. Lateral and longitudinal dose profiles have also been measured in a solid water phantom for each alternative scanning pattern to determine whether they meet clinical beam standard requirements for flatness (± 2.5%). Additionally, the secondary neutron dose equivalent was measured within an anthropomorphic phantom while the phantom was treated with a patient specific collimator and compensator inserted into the nozzle. The in-air measurements demonstrated a linear reduction in secondary neutron dose equivalent as the area of the scanning pattern decreased while the area of the treatment field remained constant. A similar linear reduction was consistently observed for different incident proton beam energies having ranges in water of 6 cm, 16 cm, and 27 cm and for changes in the size of the patient collimator. Reducing the square-shaped pattern to fit the patient collimator reduced the secondary neutron dose equivalent by approximately 20%, and circular-shaped patterns scanned in a spiral reduced the secondary neutron dose equivalent by an additional 15% for a total reduction of 35%. In combination these results determine the patient collimator as a major contributor of secondary neutrons to points distant from the target volume. The in-phantom measurements demonstrated secondary neutron dose equivalent could be reduced by optimizing the scanning pattern for the proton beam. For the square and spiral scanning patterns studied, the secondary neutron dose equivalent was decreased by approximately 30%. However a single-ring circular-shaped scanning pattern was able to reduce the neutron dose equivalent by an additional 65%