The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P CTD ) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N NTD ) as a P binding domain. A biochemical characterization of the P CTD and molecular modeling of the N NTD allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P CTD interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.H uman respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (7). It infects close to 100% of infants within the first 2 years of life and is the main cause of bronchiolitis. Also, bovine respiratory syncytial virus (BRSV), which is very similar to its human counterpart, is a major cause of respiratory disease in calves, resulting in substantial economic losses to the cattle industry worldwide (49). RSV belongs to the genus Pneumovirus of the family Paramyxoviridae and the order Mononegavirales (7). The viral genome consists of a nonsegmented ϳ15-kb RNA of negative polarity which encodes 11 proteins. As for all the members of the Mononegavirales, the genomic RNA of RSV is always tightly bound by the viral nucleoprotein (N) and maintained as a helical N-RNA ribonucleoprotein (RNP) complex (9). The RNP is used as the template for transcription and replication by the RNAdependent RNA polymerase (RdRp) complex, consisting of L (large protein) and its cofactor P (phosphoprotein). Whereas N, P, and L are sufficient to mediate viral replication (15, 50), transcription activity requires L, P, and the M2-1 protein, which functions as a processivity polymerase cofactor (8).The specific recognition of the viral N-RNA matrix by the RdRp constitutes a prerequisite for vir...

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618 Background: Muscle metastases (MM) are among rare secondary locations in renal cell carcinoma (RCC). Current guidelines do not provide specific advice on the management of these patients. There is a lack of scientific literature on the subject, mainly based on case reports or small retrospective monocentric cohorts. To date, therefore, there remains uncertainty about the clinical history, prognosis, and appropriate management of patients with MM. Methods: ARTEMIS is an ambispective national French multicenter, non-interventional study. It was opened to patients with metastatic RCC who had MM. The study was designed to assess overall survival (OS), progression-free survival (PFS), describe diagnostic and treatment modalities, and treatment-related serious adverse events in case of local treatments (TR-SAEs). Results: Median follow-up was 74.4 months IC95% [38.7-84.1]. The 146 enrolled patients were 73.6% male, with a median age of 57.6 years at diagnosis. They were initially diagnosed with stage IV RCC for 40.4% of them and had a favorable (36.2%), intermediate (45.7%), or poor (18.1%) IMDC risk score at the onset of metastases. Initially, metastases were mainly located in the lungs (55.5%), lymph nodes (26.7%), and bones (24.0%). Patients had a history of partial or total nephrectomy in 78.8% of cases. There were 30.0% of patients with synchronous MM. The median times from initial diagnosis or metastatic status to MM discovery were 25.5 [16.8-35.7] months and 8.4 [5.0-13.5] months, respectively. The majority (69.0%) of MM were discovered on conventional injected CT scans, whereas 8.3% were diagnosed clinically. Survival data are displayed on the table. Thirty-seven (25.3%) patients received local treatment of their MM which consisted of: external radiotherapy (48.6%), surgery (27.0%), cryotherapy (27.0%), stereotactic radiotherapy (5.4%), embolization (2.7%). Among them, local treatments prevented local relapse of MM in 28 patients (75.7%). Local TR-SAEs were seen in two patients (5.4%). Conclusions: ARTEMIS is the largest published cohort describing the disease history of patients with RCC suffering from MM. To our knowledge, this is the first study reporting the diagnostic and treatment modalities, also survival data. Despite its low incidence, the issue of these patients is not so rare in physicians’ practice. This work thus allows a greater understanding of clinical features and treatment of this pathology. [Table: see text]

430 Background: Primary urethral cancer (PUC) is a rare and heterogeneous malignancy. Due to its scarcity, little data are available on the optimal treatment sequence and survival, especially in metastatic patients. Methods: Data from patients diagnosed with a PUC between 01/01/2000 and 12/31/2018 were retrospectively collected from nine French referral centers. Survival rate were estimated with the Kaplan-Meier method and prognostic factors were analyzed using the Cox proportional hazards model and the log-rank test. In order to increase the statistical power of survival analysis in the metastatic stage, patients with synchronous and metachronous metastatic disease were pooled. Results: We identified 44 (62%) males and 27 (38%) females with a PUC. The most frequent histological types were urothelial carcinomas (40.0%), squamous cell carcinomas (34.3%) and adenocarcinomas (14.3%). Twenty-five (35.2%) patients were diagnosed at a localized stage (≤ T2, N0, M0); 35 (49.3%) at a locally advanced stage (≥ T3 or ≥ N1, M0) and 11 (15.5%) at a distant stage (M1). Twenty-seven patients had a metachronous metastatic cancer. Multimodality therapy was used in 24.0% and 57.1% of the patients with a localized and locally advanced disease, respectively. In the entire cohort, median overall survival (OS) was 52.5 months (IC95% 32.2 – 64.1) and stage at diagnosis was a predictor of OS (p < 0.0001). For the 60 patients with a non-metastatic disease, 39 (65%) had a recurrence or were dead and the median disease-free survival (DFS) was 21.2 months (IC95% 16.8 – 34.5). Nodal involvement was the only factor associated with DFS (HR: 2.03, p = 0.0390). Multimodal treatment compared with unimodal treatment was not significantly associated with DFS (HR: 1.22, p = 0.5419). Regarding survival of the 38 metastatic patients, the median OS was 15.2 months (IC95% 8.2 – 23.5) and the median progression-free survival was 6.4 months (IC95% 4.4 – 9.8). Conclusions: This retrospective study showed an important heterogeneity in terms of histology, stage at diagnosis, and treatment of PUC. In this cohort, the multimodal approach did not show any improvement in survival of non-metastatic patients. This study is one of the few to describe the survival in metastatic patients.