Viral PIC-pocketing: RSV sequestration of translational preinitiation complexes into bi-phasic biomolecular condensates.

Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Here, we report a strategy whereby the orthopneumoviruses sequester various components of the translational preinitiation complex machinery into viral inclusion bodies to facilitate translation of their own mRNAs-PIC-pocketing. Electron microscopy of respiratory syncytial virus (RSV)-infected cells revealed bi-phasic organization of IBs, specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy, combined with fluorescence in situ hybridization, we showed that the observed bi-phasic morphology represents functional compartmentalization of the inclusion body and that these domains are synonymous with the previously reported inclusion body-associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as components of eukaryotic translation initiation factors (eIF), eIF4F and eIF3, and 40S complexes involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates that the majority of viral mRNA translation occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however, interestingly, ribopuromycylation assays showed no changes to global levels of translation. The mechanistic basis for this pathway was subsequently determined to involve the viral M2-1 protein interacting with eIF4G, likely to facilitate its transport between the cytoplasm and the separate phases of the viral inclusion body. In summary, our data show that these viral organelles function to spatially regulate early steps in viral translation within a highly selective bi-phasic biomolecular condensate. IMPORTANCE: Respiratory syncytial viruses (RSVs) of cows and humans are a significant cause of morbidity and mortality in their respective populations. These RNA viruses replicate in the infected cells by compartmentalizing the cell's cytoplasm into distinct viral microdomains called inclusion bodies (IBs). In this paper, we show that these IBs are further compartmentalized into smaller structures that have significantly different density, as observed by electron microscopy. Within smaller intra-IB structures, we observed ribosomal components and evidence for active translation. These findings highlight that RSV may additionally compartmentalize translation to favor its own replication in the cell. These data contribute to our understanding of how RNA viruses hijack the cell to favor replication of their own genomes and may provide new targets for antiviral therapeutics in vivo.

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins.

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.

BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein Production to Inhibit Cell-Cell Fusion.

The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus-cell and cell-cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell-cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell-cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell-cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype.

A cooperative network of trained sites for the conduct of a complex clinical trial: a new concept in multicenter clinical research.

BACKGROUND: The purpose of this report is to present a model of physicians in full-time clinical practice participating as investigators in multicenter clinical trials, sponsored by a pharmaceutical or medical device company. METHODS: This gas-exchange substudy was conducted as a pilot study to establish the feasibility of the 10-member EXERcise testing group of the Duke University Cooperative Cardiovascular Society (EXERDUCCS) consortium to perform a complex multicenter trial using cardiopulmonary exercise testing. An active interchange of information was established involving the principal investigator for the substudy, a dedicated full-time project coordinator, a medical director of the overall EXERDUCCS network site, the project coordinator for the sponsor, and all the participating EXERDUCCS investigators and coordinators. RESULTS: The sponsor set as a goal of enrollment of 6 subjects per site, and 8 of the 10 sites met this goal. As a result of the successful enrollment and completion of the study and substudy by the EXERDUCCS sites, the sponsor subsequently increased the payment stipends to the sites to compensate for the extra work and expense incurred. CONCLUSIONS: This cooperative experience accomplished several goals: (1) it allowed a complex clinical trial to be successfully completed in a time frame which would not have been possible using only single unconnected sites; (2) it educated the physician-investigators (and their personnel) in exercise cardiopulmonary; and (3) it prepared the sites for future clinical trials involving this methodology.

The practice of certified community health CNSs.

PURPOSE/OBJECTIVES: This study explored the practice of clinical nurse specialists (CNSs) certified in Community Health nursing in the United States and described demographic and employment characteristics and perspectives about professional practice. METHODS: The survey method was used. Of the 209 Community Health CNSs certified by American Nurses Credentialing Center (ANCC) invited to complete the investigator-designed mail questionnaire, 111 (53%) returned a completed questionnaire. The questionnaire contained 27 items about employment, income, years in practice, certification, career satisfaction, and educational preparation, and asked participants to indicate the fit between the Community Health CNS role and the traditional CNS subroles model described by the American Nurses Association (ANA) (The Role of the Clinical Nurse Specialist, 1986) and the updated National Association of Clinical Nurse Specialists (NACNS) CNS practice model (Statement on Clinical Nurse Specialist Practice and Education, 1998). Content validity was established by Community Health CNS reviewer feedback. ANALYSIS: Quantifiable data were tallied and analyzed using standard spreadsheet computer software. Qualitative data were summarized for content themes. FINDINGS: The majority of participants were white, middle-aged females who reported being satisfied with their careers as Community Health CNSs. Most indicated that they were respected by colleagues, that they had been adequately prepared by their education, and that their current work made good use of their education and expertise. When asked to identify, by percentage of effort, the fit between their job responsibilities and the traditional subroles model of practice, the mean of reported fit was as follows: educator, 35%; administrator/leader, 22%; clinician, 21%; consultant, 14%; and researcher, 8%. The fit between job responsibilities and the spheres of influence in the NACNS model of practice was reported to average 39% for patient/client, 35% for organization/network, and 25% for nurses/ nursing practice. CONCLUSIONS: Community Health CNS is a viable specialty practice with long-term career options. The subrole functions-described by ANA-of clinician, educator, administrator/leader, consultant, and to a lesser extent researcher apply to the role. The more intergraded updated model offered by NACNS also fits Community Health CNS practice with more emphasis on patient/client and organization/ network spheres than on nurses/nursing practice sphere. IMPLICATIONS: Schools of nursing should continue to offer the Community Health CNS programs and incorporate both the traditional functions and newer practice model into their curricula, with a greater emphasis on diversity of students to help ensure a more diverse CNS population. Further research is needed to explore the outcomes of Community Health CNS practice and the factors that contribute to role satisfaction.