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

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses

DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV.

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an ∼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development

Atomic structures and deletion mutant reveal different capsid-binding patterns and functional significance of tegument protein pp150 in murine and human cytomegaloviruses with implications for therapeutic development.

Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a "Λ"-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a "Δ"-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies

Regional differences of physical fitness and overweight and obesity prevalence among college students before and after COVID-19 pandemic since the “double first-class” initiative in China

IntroductionPhysical fitness has been widely recognized as a powerful marker of health in children and adolescents, and it negatively affected by the COVID-19 pandemic. The construction of world-class universities and first-class disciplines, known as the “Double First-Class” Initiative (DFC), is a major commitment made by the Chinese government to adapt to changes in the educational environment, both domestically and internationally, in order to promote the development and practice of international higher education. The aim of the study was to look deep into the regional differences of physical fitness and overweight and obesity prevalence among college students before and after the COVID-19 pandemic since the DFC.MethodsThe original physical fitness parameters of students from 10 DFC universities and colleges in Central South China were downloaded from the official website of Chinese National Student Physical Fitness Database (CNSPFD) and then divided into 3 groups based on the pandemic periods: pre-pandemic (2019), the first year after pandemic outbreak (2020), and the second year after pandemic outbreak (2021). All the data were stored in Excel 2010, analyzed by SPSS 17.0, and plotted with ArcGIS 10.4.ResultsThe total “fail” percentage (from 9.19% in 2019 to 12.94% in 2021) and the prevalence of overweight and obesity in boys (from 22.53 to 29.25% in 2021) exhibited a continuous increase year by year, and among all the physical fitness indicators the score of strength in boys and endurance quality in all individuals were the lowest in overweight and obesity groups. Students with ‘fail’ rate developed from northern and northeastern province to southern areas from 2019 to 2021. For grade 2019th, overweight and obesity students who also failed the test had covered nationwide and the most affected areas including northeast, east, as well as central north in senior year. The distribution of overall fitness assessments in Hubei province was in accordance with the national data, and the overall scoring growths in both class of 2021st and 2022nd were measured with a negative increase (p < 0.01).ConclusionThe government and related functional departments should take into consideration the student regional sources, especially in western and northeast regions of China, and school polices and physical education (PE) teachers should pay more attention to put training efforts on endurance for all adolescents and strength for boys and the group of overweight and obesity who also failed in the standard test, when designing specific interventions to promote physical health and counteract the negative effects of COVID-19 pandemic in college students

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus

双链RNA病毒是病毒中最大的类群，其中轮状病毒作为双链RNA病毒家族最知名的病毒之一，每年引起接近百万的新生儿死亡，而单链RNA病毒虽然没有双链RNA病毒那么多，但是却包含了许多很有名的病毒：HIV，埃博拉病毒，小核糖核苷酸病毒（包括甲肝病毒HAV、肠道病毒、脊髓灰质炎病毒、口蹄疫病毒、感冒病毒等），SARS病毒，丙肝病毒HCV等等。而且与双链RNA病毒不同的是，单链RNA病毒不会把它们的基因组包裹到预制的外壳蛋白中，而是利用基因组共同装配壳体，关于这个共同组装的过程，科学家们了解的很少。最新研究中，研究人员获取了单链RNA病毒：大肠杆菌噬菌体MS2的冷冻电镜结构（分辨率为3.6Å），并追踪了80%病毒基因组结构，从而发现了这种病毒的壳体共同组装过程中的分子机制，这将为解析核蛋白复合物与病毒起源之间的联系提供了重要的信息。【Abstract】Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid1–3 , single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome4–7 ; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’(F-pilus)8 ; it was the first fully sequenced organism9 and is a model system for studies of translational gene regulation10,11,RNA–protein interactions12–14 , and RNA virus assembly15–17 .Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T=3 icosahedral lattice18,19 . The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection8 ,but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryoelectron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome,built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′end of the genome interacts extensively with the maturation protein,which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.This project was supported in part by grants from the National Institutes of Health (GM071940, DE025567, DE023591, CA177322 and AI094386) and National Science Foundation (DMR-1548924). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines (supported by UCLA and by instrumentation grants from the NIH (1S10OD018111, 1U24GM116792) and NSF (DBI-1338135))

Membrane insertion of—and membrane potential sensing by—semiconductor voltage nanosensors: Feasibility demonstration

We developed membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. We provide here a feasibility study for their utilization. We use a rationally designed peptide to functionalize the nanosensors, imparting them with the ability to self-insert into a lipid membrane with a desired orientation. Once inserted, these nanosensors could sense membrane potential via the quantum confined Stark effect, with a single-particle sensitivity. With further improvements, these nanosensors could potentially be used for simultaneous recording of action potentials from multiple neurons in a large field of view over a long duration and for recording electrical signals on the nanoscale, such as across one synapse