Antiviral activity of intracellular nanobodies targeting the influenza virus RNA-polymerase core.

Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin ß RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development.

Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals.

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

The Influenza Virus RNA-Polymerase and the Host RNA-Polymerase II: RPB4 Is Targeted by a PB2 Domain That Is Involved in Viral Transcription.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5'-end of nascent host mRNA.

Functional Results of Cervical Rib Resection for Thoracic Outlet Syndrome: Impact on Professional Activity.

BACKGROUND: The aim of this study is to report long-term functional results following cervical rib (CR) resection for thoracic outlet syndrome (TOS). METHODS: This monocentric study included all cases of resection of CR for TOS performed between January 2004 and December 2016. Data were retrospectively collected from the hospital electronic database including preoperative symptoms and the evaluation of occupational well-being, intraoperative data, and early clinical evaluation and occupational well-being during the postoperative period. Patients were categorized as neurogenic TOS (NTOS), arterial TOS (ATOS), arterial and neurogenic TOS (ANTOS), venous TOS (VTOS), or asymptomatic according to preoperative evaluation. We evaluated the improvement in work life between the preoperative and the postoperative period. Further assessment was a negative Roos or elevated arm stress test (EAST) during the postoperative period. RESULTS: Thirty-three patients with a median age of 38.5 years (30-46) were included. Thirty-six procedures were performed: 33% to treat ATOS (12/36), 39% for NTOS (14/36), 19% for ANTOS (7/36), 3% for VTOS (1/36), and 6% (2/36) for asymptomatic lesions. There were 9 cases of subclavian artery aneurysms leading to additional arterial repair. Due to distal embolization, a cervical sympathectomy was associated in 5 procedures. First rib resection was associated in 4 procedures (11%) and C7 transverse process resection was performed in 15 procedures (42%). The technical success rate was 100% and intraoperative complications were observed in 4 patients (11%) with favorable postoperative outcomes. During the early postoperative period, 3 Claude Bernard-Horner's syndrome and 1 asymptomatic subclavian dissection were detected. Late complications included 2 bypass thromboses (6%) at 6 weeks and 16 months. Postoperative EAST improved in 16 limbs (44%). Prior to the procedure, only 27% (9/33) patients had normal work lives. After the procedure, 64% (21/33) of patients were able to return to their normal work activity. CONCLUSIONS: CR resection for TOS seems to be a safe procedure leading to good short- and long-term clinical results with a favorable impact on recovering a normal work life in these young patients.

Femoral Bifurcation Endarterectomy with Transection-Eversion of the Superficial Femoral Artery: Technique and Results.

BACKGROUND: We evaluated the results of femoral bifurcation endarterectomy using the eversion technique with transection of the superficial femoral artery (femoral bifurcation endarterectomy with eversion [FBEE]). METHODS: We included all patients who underwent a femoral revascularization using the eversion technique, with or without antegrade or retrograde revascularization, from January 2006 to December 2015. Data were retrospectively collected. Primary and primary assisted patency (PAP) of the femoral bifurcation were analyzed. Secondary outcomes were 30-day postoperative complications. RESULTS: A total of 129 patients (143 limbs) underwent consecutive FBEE (86.8% men, with a mean age of 69.7 years). Patients presented with claudication (93, 65%) and critical ischemia (46, 32.2%). Primary patency was 96.3%, 94.6%, and 93% at 1, 2, and 5 years, respectively. PAP was 99% at 3 time points. Reintervention was necessary in 8 patients during follow-up. The 30-day mortality was 0.7% (1 patient), and the access complication rate was 18.8% (n = 27), of which only 2.8% (n = 4) were major complications. CONCLUSIONS: This retrospective study confirmed the efficiency and the reproducibility of this technique for the treatment of femoral bifurcation lesions. This technique allowed treating extensive atherosclerotic lesions of the deep femoral artery and may be associated with antegrade and retrograde revascularizations.