A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins.

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein.

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by three to four different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced coassembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

Impact of telehealth on health care in a multiple sclerosis outpatient clinic during the COVID-19 pandemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has precipitated expansion of telemedicine in outpatient management of chronic diseases including multiple sclerosis (MS). Studies conducted pre-pandemic, when telehealth was an alternative to in-person consultations, represent a different setting to current practice. The aim of this study was to assess the impact of telehealth on MS outpatient care in a tertiary metropolitan hospital in Melbourne, Australia during the COVID-19 pandemic. METHOD: From March-December 2020, patients and clinicians in the MS outpatient clinic were surveyed regarding their attitudes towards telehealth. Scores on the Expanded Disability Status Scale (EDSS) from telehealth and face-to-face appointments during the study period were compared to scores from face-to-face consultations before and after this period. Medical records were reviewed to compare management decisions made during telehealth versus face-to-face consultations. Diagnoses and treatment of MS relapses were compared to 2019. RESULTS: Telehealth was used in 73% of outpatient appointments. Patient satisfaction was generally high. Patients and clinicians preferred face-to-face consultations but were willing to use telehealth longer term. Overall, there were no significant delays in identifying patients experiencing disability worsening via telehealth, but EDSS increase was recorded in more face-to-face than telehealth appointments particularly for those with lower baseline disability. Disease-modifying therapy commencement rates were similar, but symptomatic therapy initiation and investigation requests occurred more frequently in face-to-face visits. Comparable numbers of MS relapses were diagnosed and treated with corticosteroids in 2019 and 2020. CONCLUSIONS: Patient satisfaction with telehealth was high, but both clinicians and patients preferred in-person appointments. Telehealth implementation did not lead to high rates of undetected disability worsening or undiagnosed acute relapses, but telehealth-based EDSS assessment may underestimate lower scores. Treatment inertia may affect some management decisions during telehealth consultations. Telehealth will likely play a role in outpatient settings beyond the COVID-19 pandemic with further studies on its long-term impact on clinical outcomes required.

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids.

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here, we use biophysical tools to study N-protein interactions with oligonucleotides of different lengths, examining the size, composition, secondary structure, and energetics of the resulting states. We observe the formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant change in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates.

Fast and safe: Optimising multiple sclerosis infusions during COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic challenges multiple sclerosis services to be innovative in delivering infusible therapies. To reduce time in clinical settings, and potential staff or space losses, we implemented rapid infusion protocols for selected patients. OBJECTIVE: To analyse the rate of infusion related reactions and patient experience of rapid infusions of natalizumab and ocrelizumab. To document time reduction patients spent in clinical settings during the COVID-19 pandemic. METHODS: Patients with prior exposure to at least three natalizumab or two 300mg ocrelizumab infusions were approved for rapid protocols. A retrospective audit and survey were completed. RESULTS: We analysed 269 rapid natalizumab infusions and 100 rapid ocrelizumab infusions. Infusion related reactions during the natalizumab or ocrelizumab infusions occurred in two patients (1.52%) and eight patients (8%), respectively. All infusion related reactions were mild to moderate and did not require infusion discontinuation. No infusion reactions occurred during the post-infusion observation. Patient experience was positive. CONCLUSION: Frequency or severity of infusion related reactions in rapid infusions were no different compared to published data. In the setting of COVID-19, pandemic rapid infusion protocols could potentially save hospital resources and limit patient exposure to a high-risk clinical setting while still maintaining ongoing treatment of multiple sclerosis.

Evaluating the perspective of patients with MS and related conditions on their DMT in relation to the COVID-19 pandemic in one MS centre in Australia.

OBJECTIVE: Patients with Multiple Sclerosis (MS) and on disease modifying therapies (DMTs) that can be immunosuppressive or immunomodulatory form a special group where risk of continuation of DMT needs to be taken into account with risk of contracting Covid-19. This concept can pose a degree of anxiety for patients as well as neurologists. We aimed to evaluate patient perspectives regarding the use of Natalizumab and anti-CD20 therapies (Rituximab and Ocrelizumab) in the context of the COVID-19 pandemic. METHODS: cross-sectional study conducted via voluntary survey filled in by patients with MS and related disorders receiving their infusional treatment in one MS centre in Australia, exploring their concerns regarding their therapy, their therapy and COVID-19, precautions undertaken in response to the pandemic, and factors impacting their decision-making. RESULTS: 170 patients completed the survey. Of patients on Natalizumab, the majority had either no or mild concern regarding their DMT and COVID-19, and of patients on B-cell depleting therapies, again, the majority had no or mild concern, though a slightly higher proportion had a moderate level of concern. Asked to delineate their concerns, an increased risk of contracting COVID-19 was more commonly conveyed than MS-specific factors or poor outcomes pertaining to COVID-19 if contracted, by patients in both groups. Conversely, being invited to specifically consider the possibility of contracting COVID-19 or experience a relapse of MS, almost half of the cohort rated both of equal of concern. More than half of the cohort were self-isolating more stringently than general government advice and government-related resources followed by information provided by patient's neurologist where the commonest means of information to guide decision making. CONCLUSIONS: Whilst a large proportion of patients had some concern regarding the impact of their DMT on COVID-19, whether on their risk of contracting COVID-19 or a theoretical risk for more severe disease, the overall level of concern in most cases was at most mild. Patients on B-cell depleting therapies were more inclined to express a higher level of concern. A similar concern was ascribed to a risk of a relapse or worsening MS symptoms compared to the risk of contracting COVID-19. Such attitudes may underscore a willingness of patients to continue their DMT where benefits outweigh risks during future phases of the COVID-19 pandemic.