Structures of the promoter-bound respiratory syncytial virus polymerase.

The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs1. The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome2. However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV.

Increasing Follow Up With Primary Care Providers After Emergency Department Visits in Older Adults.

PURPOSE: To increase follow up with the primary care team via telephone outreach within 3 days of emergency department (ED) discharge to schedule a follow-up visit within 14 days. Secondary aims included: identifying high utilizers of the ED (defined as more than three ED visits within 6 months), reinforcing discharge instructions from the ED via nursing education on telephone follow ups, and identifying the reasons patients used the ED. METHOD: Baseline data were gathered retrospectively by reviewing charts of patients discharged from the ED. Charts were reviewed from a biweekly automated report, and RNs initiated follow-up phone calls to patients discharged from the ED, offering appointments and providing pertinent nursing education. RESULTS: Primary care follow ups after ED discharges increased from 38% to 71% over 10 months with the new nurse-led workflow. Patients to whom the RN outreached to were more likely to attend their follow-up appointments. However, a 14-day follow-up appointment with the primary care provider (PCP) showed no significant difference in ED revisits or hospital admissions. CONCLUSION: Follow up after ED discharge led to increased coordination of care. Nurses provided education about chronic conditions and reiterated discharge instructions that might have been unclear to patients in the ED. Further studies are needed to analyze the effect of follow up with the PCP on ED utilization and hospital admissions. [Journal of Gerontological Nursing, 50(3), 33-39.].

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

Converting patients from warfarin to non-vitamin K antagonist oral anticoagulants.

BACKGROUND: Non-vitamin K antagonist oral anticoagulants (NOACs) are favorable in stroke prevention for geriatric patients with nonvalvular atrial fibrillation versus warfarin. These anticoagulants do not require international normalized ratio (INR) monitoring and have lower food/drug interactions. In addition, NOACs have risk reduction in bleeding and all-cause mortality compared with warfarin. LOCAL PROBLEM: At a geriatric primary care practice, two registered nurses manage 88 patients on warfarin for INR monitoring. Nurse practitioners (NPs) provide oversight for warfarin titration after abnormal results. The goal of this quality-improvement project was to decrease the time spent monitoring patients on warfarin. METHODS: Primary care providers and cardiologists of patients on warfarin were contacted to gain approval of transition to a NOAC. The NP reviewed patients' renal function and the indication for anticoagulation and then created a list of eligible patients for transition. INTERVENTIONS: Patients eligible for transition to NOACs were contacted for their consent. The transition process included stopping warfarin, ordering apixaban, ordering INR level, educating about starting apixaban, and coordinating appropriate follow-up. RESULTS: Of 88 patients on warfarin, 21 were eligible for conversion from warfarin to apixaban. Of these 21 patients, 66% ( n = 14) consented to the conversion. Of those who were not converted to apixaban, five declined due to cost and two were lost to follow-up. CONCLUSION: There was a reduction in nurses' monthly monitoring of patients on warfarin by 22%. Transition to NOAC was not only beneficial for patient safety and efficacy but also reduced nursing clinical time for anticoagulation encounters.

Cryo-EM structure of the respiratory syncytial virus RNA polymerase.

The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.