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.

Addition of Postoperative Radiation Therapy After Preoperative Chemotherapy and Surgery in Patients With Locally Advanced Endometrial Cancer Is Associated With Improved Outcomes.

Purpose: Our purpose was to examine outcomes of patients with locally advanced endometrial cancer who undergo neoadjuvant chemotherapy followed by surgery (PreCT) with/without postoperative adjuvant radiation therapy. A secondary analysis of down staging and margin clearance was made with reference to those receiving upfront surgery and then adjuvant chemotherapy (PostCT). Methods and Materials: The National Cancer Database was queried for FIGO (The International Federation of Gynecology and Obstetrics) stage III/IV locally advanced endometrial cancer cases who underwent definitive surgery from 2010 to 2016 and received chemotherapy as part of their treatment. Cases were classified into 2 cohorts: preoperative chemotherapy +/- postoperative chemotherapy cohort (PreCT) and postoperative chemotherapy cohort (PostCT) for reference for margin assessment. Cases who received preoperative radiation therapy were excluded while those who received postoperative radiation were included in the analysis. Primary endpoints were overall survival (OS), surgical margin status, rate of downstaging, and effect of adjuvant radiation therapy on OS among the PreCT cohort. Univariable (UVA) and multivariable (MVA) Cox regression analyses were performed. Results: A total of 13,369 cases were identified with 1059 in PreCT and 12,310 in PostCT cohorts. PreCT had lower OS than PostCT (UVA: hazard ratio [HR], 2.18; P < .001; MVA: HR, 1.873; P < .001). PreCT cases with negative margins, who presumably had unresectable tumors initially, also had worse OS compared with PostCT with negative margins (UVA: HR, 2.20; P < .001; MVA: HR, 1.84; P < .001); however, PreCT with negative margins had similar survival to PostCT with positive margins (UVA: HR, 0.825; P < .001; MVA: P = .885). The addition of radiation after surgery in the PreCT cohort was associated with improved survival (5-year OS 20.5% compared with 50%, respectively; UVA: HR, 0.450; P < .001; MVA: HR, 0.337; P < .001). Although fewer cases in PreCT had negative margins compared with PostCT (72% compared with 84%, P < .001), approximately 19% of cases in PreCT had lower pathologic T-stage compared with clinical T-stage and 11% had lower N-stage. Conclusions: Neoadjuvant chemotherapy was given in cases with worse oncologic prognostic factors, many of whom were likely unresectable at outset, compared with those who received postoperative chemotherapy. Although neoadjuvant chemotherapy is associated with tumor downstaging, survival is lower than with primary surgery probably because of these baseline differences. The addition of adjuvant radiation after surgery in cases who received preoperative chemotherapy is associated with improved survival.

Change in image-guided planning strategies over time impacts oncologic and survival outcomes for intracavitary cervical cancer brachytherapy.

PURPOSE: Intracavitary cervical brachytherapy (BT) has transitioned from a two-dimensional nonvolumetric (NV) dosimetry system to three-dimensional computed tomography (CT) and/or magnetic resonance imaging (MRI)-based planning techniques. The purpose of this study is to retrospectively evaluate the relative improvements in image-guided planning strategies over time with regards to dosimetry, survival, and toxicity. METHODS AND MATERIALS: A single site retrospective review of 95 locally advanced cervical cancer patients treated with concurrent chemoradiation and high dose rate BT from 2009 to 2016 were divided into three BT planning groups: point-A based NV dosimetry using CT imaging (n = 37), CT-based volumetric dosimetry (n = 33), and MRI-based volumetric dosimetry (n = 25). Overall survival (OS), progression free survival (PFS), and pelvic control (PC) at 5 years were plotted using Kaplan-Meier curves. Univariate and multivariate (MVA) cox proportional-hazards models calculated hazard-ratios (HZ). Finally, acute and late grade 3-4 toxicities were compared between the cohorts. RESULTS: Both MRI and CT had significantly less D2cc to bowel (p < 0.001) and sigmoid (p < 0.001) compared to NV-based planning. On MVA, age (<60 vs. ≥60 years) was significant for worse 5-year OS (HZ: 2.48) and PC (HZ: 5.25). MRI, with NV as the reference, had significantly improved 5-year OS (HZ: 0.26), PFS (HZ: 0.34) and PC (HZ: 0.16). There was no significant difference in grade ≥3 toxicities between the cohorts. CONCLUSIONS: CT and MRI-based 3D planning had significantly less D2cc to bowel and sigmoid. MRI-based planning had significant improvement in 5-year OS, PFS, and LC compared to NV on MVA.

Correlating Quantitative Para-Aortic Lymph Node Computed Tomography Parameters With Fluorodeoxyglucose Positron Emission Tomography for Cervical Cancer Staging: Possible Solution for Resource Constrained Countries.

OBJECTIVE: The aim of the study was to determine the most accurate quantitative morphological parameters on computed tomography (CT) that correlate with fluorodeoxyglucose (FDG)-avid para-aortic nodes (PANs) in patients with cervical cancer. METHODS: A single-institution retrospective evaluation was performed of women with cervical cancer who underwent pretreatment positron emission tomography (PET)/CT and radiotherapy therapy planning CT between 2009 and 2020. A node-by-node correlation between pretreatment CT and PET/CT was performed for the reference standard of FDG avidity for short- and long-axis diameters, volume, and long-/short-axis ratio (L/S). The FDG-avid PANs were defined as PET-positive and non-FDG-avid PANs from patients without PET-determined PAN metastasis were defined as PET negative. Area under the receiver operator curve was calculated to access diagnostic accuracy of the different quantitative parameters. RESULTS: A total of 94 women (mean age ± standard deviation, 52 ± 13 years) with cervical cancer were included. Forty-seven patients had PET-positive PANs (181 PET-positive PANs) and 47 patients had no PET-positive PANs (141 PET-negative PANs). The area under the receiver operator curve for volume (0.945) was greater ( P < 0.001) than that of short axis (0.895), long axis (0.885), and L/S (0.583). At a specificity set point of 0.90 (127/141 PANs), the cutoff for volume was 0.443 cm 3 or greater (0.85 sensitivity [154/181 PANs]; 95% confidence interval, 0.83-0.93) and for short-axis diameter was 5.9 mm or greater (0.75 sensitivity [135/181 PANs]; 95% confidence interval, 0.68-0.81). CONCLUSIONS: Para-aortic lymph node volume demonstrated that improved node-by-node correlation between CT and PET/CT compared with short-axis diameter, long-axis diameter, and L/S and is an alternative to improve detection of PAN suspicious of metastatic diseases in locations without access to PET/CT.

Updating and Optimizing Anatomic Atlases for Elective Radiation of Para-Aortic Lymph Nodes in Cervical Cancer.

PURPOSE: Previous studies have proposed 2 different contouring guidelines for the prophylactic radiation of para-aortic lymph nodes (PANs) for locally advanced cervical cancer. Because PAN-mapping atlases in current literature are limited to small patient samples and nodal populations, we updated the PAN atlas with a large data set of positron emission tomography (PET)-positive PANs on PET/computed tomography (CT) from patients with cervical cancer. METHODS AND MATERIALS: We identified 176 PET-positive PANs on pretreatment PET/CT of 47 patients with diagnosed International Federation of Gynecology and Obstetrics stage IB to IVA cervical cancer. PANs were classified as left-lateral para-aortic (LPA), aortocaval (AC), or right paracaval (RPC). PAN clinical target volume (CTV) contours were drawn for all patients based on previously published guidelines by Takiar (CTV-T) and Keenan (CTV-K) and nodal volumetric coverage was assessed. RESULTS: We identified 94 LPA nodes (54%), 71 AC nodes (40%), and 11 (6%) RPC nodes. CTV-T had improved nodal center coverage of 97.6% compared with 85.0% for CTV-K (P < .001). Nodal center coverage for CTV-K and CTV-T (with corresponding PAN) were 79 (84.0%) and 93 (99.0%) LPA nodes (P = .001), 64 (90.1%) and 68 (95.8%) AC nodes (P = .221), and 5 (45.5%) and 9 (81.8%) RPC nodes (P = .134), respectively. Additionally, our updated PAN atlas identified nodal centers anterior to the aorta and inferior vena cava that are not covered by CTV-T but covered by CTV-K due to the 10 mm anterior aortic expansion of CTV-K. CONCLUSIONS: We have updated the PAN anatomic map of 176 PET-positive nodes from 47 patients and demonstrated that CTV-T has significantly better PAN coverage over CTV-K for posterior LPA and retrocaval regions for our data set. Additionally, we suggest a modification that includes a blend of CTV-T and CTV-K to provide optimal coverage for the mapped nodes anterior to the great vessels in our data set.

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.

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.