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The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress–budding at the inner nuclear membrane–is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

60 APPLIED LIFE SCIENCES↗

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

membrane curvature↗

User Guide for the Los Alamos National Laboratory Fallout Calculator (V.1.1)

The Los Alamos National Laboratory Fallout Calculator is a Java™ application that can calculate the dose from many nuclear detonations producing fallout. The fallout calculations are performed for one or multiple selected winds. The winds are from the years 2000 to 2010, for all 12 months, and for the 1 st , 10 th , and 20 th of each month. The winds are available from 0 meters to 50,000 meters altitude MSL. This Fallout Calculator performs ideal calculations. There is no terrain. The ground is perfectly flat at sea level. There is no rain that would cause radioactive hot spots. The wind is historical, not a current wind. Also, the wind is fixed in time. It does not vary over the 24 hours it takes the fallout to arrive on the ground. Therefore, this calculator is suitable for operational type studies. For instance, it can be used to calculate the relative difference in fallout patterns from different possible nuclear laydowns. It can be used to understand how frequently fallout would be likely to occur in a particular part of the US. It could be used to develop plans for how to protect the population from a nuclear attack. However, it should not be used to predict the dose in a city from an actual laydown, or to predict safe corridors for the population to use to egress from areas with high fallout.

45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEF↗

Portal Monitoring Considerations for Treaty Verification Applications

Portal monitoring is one method in a suite of options for treaty verification and can assist in verification of treaty accountable items (TAIs). Portal monitor technology spans a wide range of sensors, including radiation detectors, break-beams, or weight sensors. For radiation-detecting portal monitors, they are able to confirm absence / presence of a radiological signature and to track TAI direction of motion. Portal monitoring uses measurements from strategically placed sensors (e.g., radiation detectors) to record the entry or egress of TAIs. The implementation of portal monitoring relies on three key elements: perimeter definition, portal locations, and sensor technology, and can be implemented in either time-bound or continuous operations depending on what is permitted by the Treaty.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗