Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome.

Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid-with triangulation number (T) equal to 16-encapsidating a tightly packed double-stranded DNA (dsDNA) genome1-3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Although this process has been studied in dsDNA phages6-9-with which herpesviruses bear some similarities-a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal-a longstanding mystery in icosahedral viruses-and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging

Postsynaptic protein organization revealed by electron microscopy.

Neuronal synapses are key devices for transmitting and processing information in the nervous system. Synaptic plasticity, generally regarded as the cellular basis of learning and memory, involves changes of subcellular structures that take place at the nanoscale. High-resolution imaging methods, especially electron microscopy (EM), have allowed for quantitative analysis of such nanoscale structures in different types of synapses. In particular, the semi-ordered organization of neurotransmitter receptors and their interacting scaffolds in the postsynaptic density have been characterized for both excitatory and inhibitory synapses by studies using various EM techniques such as immuno-EM, electron tomography of high-pressure freezing and freeze-substituted samples, and cryo electron tomography. These techniques, in combination with new correlative approaches, will further facilitate our understanding of the molecular organization underlying diverse functions of neuronal synapses

Accumulation of Dense Core Vesicles in Hippocampal Synapses Following Chronic Inactivity.

The morphology and function of neuronal synapses are regulated by neural activity, as manifested in activity-dependent synapse maturation and various forms of synaptic plasticity. Here we employed cryo-electron tomography (cryo-ET) to visualize synaptic ultrastructure in cultured hippocampal neurons and investigated changes in subcellular features in response to chronic inactivity, a paradigm often used for the induction of homeostatic synaptic plasticity. We observed a more than 2-fold increase in the mean number of dense core vesicles (DCVs) in the presynaptic compartment of excitatory synapses and an almost 20-fold increase in the number of DCVs in the presynaptic compartment of inhibitory synapses after 2 days treatment with the voltage-gated sodium channel blocker tetrodotoxin (TTX). Short-term treatment with TTX and the N-methyl-D-aspartate receptor (NMDAR) antagonist amino-5-phosphonovaleric acid (AP5) caused a 3-fold increase in the number of DCVs within 100 nm of the active zone area in excitatory synapses but had no significant effects on the overall number of DCVs. In contrast, there were very few DCVs in the postsynaptic compartments of both synapse types under all conditions. These results are consistent with a role for presynaptic DCVs in activity-dependent synapse maturation. We speculate that these accumulated DCVs can be released upon reactivation and may contribute to homeostatic metaplasticity