SAMD9L acts as an antiviral factor against HIV-1 and primate lentiviruses by restricting viral and cellular translation.

Sterile alpha motif domain-containing proteins 9 and 9-like (SAMD9/9L) are associated with life-threatening genetic diseases in humans and are restriction factors of poxviruses. Yet, their cellular function and the extent of their antiviral role are poorly known. Here, we found that interferon-stimulated human SAMD9L restricts HIV-1 in the late phases of replication, at the posttranscriptional and prematuration steps, impacting viral translation and, possibly, endosomal trafficking. Surprisingly, the paralog SAMD9 exerted an opposite effect, enhancing HIV-1. More broadly, we showed that SAMD9L restricts primate lentiviruses, but not a gammaretrovirus (MLV), nor 2 RNA viruses (arenavirus MOPV and rhabdovirus VSV). Using structural modeling and mutagenesis of SAMD9L, we identified a conserved Schlafen-like active site necessary for HIV-1 restriction by human and a rodent SAMD9L. By testing a gain-of-function constitutively active variant from patients with SAMD9L-associated autoinflammatory disease, we determined that SAMD9L pathogenic functions also depend on the Schlafen-like active site. Finally, we found that the constitutively active SAMD9L strongly inhibited HIV, MLV, and, to a lesser extent, MOPV. This suggests that the virus-specific effect of SAMD9L may involve its differential activation/sensing and the virus ability to evade from SAMD9L restriction. Overall, our study identifies SAMD9L as an HIV-1 antiviral factor from the cell autonomous immunity and deciphers host determinants underlying the translational repression. This provides novel links and therapeutic avenues against viral infections and genetic diseases.

The HIV-1 Integrase C-Terminal Domain Induces TAR RNA Structural Changes Promoting Tat Binding.

Recent evidence indicates that the HIV-1 Integrase (IN) binds the viral genomic RNA (gRNA), playing a critical role in the morphogenesis of the viral particle and in the stability of the gRNA once in the host cell. By combining biophysical, molecular biology, and biochemical approaches, we found that the 18-residues flexible C-terminal tail of IN acts as a sensor of the peculiar apical structure of the trans-activation response element RNA (TAR), interacting with its hexaloop. We show that the binding of the whole IN C-terminal domain modifies TAR structure, exposing critical nucleotides. These modifications favour the subsequent binding of the HIV transcriptional trans-activator Tat to TAR, finally displacing IN from TAR. Based on these results, we propose that IN assists the binding of Tat to TAR RNA. This working model provides a mechanistic sketch accounting for the emerging role of IN in the early stages of proviral transcription and could help in the design of anti-HIV-1 therapeutics against this new target of the viral infectious cycle.

Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase.

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner.

The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.

Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.

Upf1 is a crucial factor in nonsense-mediated mRNA decay, the eukaryotic surveillance pathway that degrades mRNAs containing premature stop codons. The essential RNA-dependent ATPase activity of Upf1 is triggered by the formation of the surveillance complex with Upf2-Upf3. We report crystal structures of Upf1 in the presence and absence of the CH domain, captured in the transition state with ADP:AlF4⁻ and RNA. In isolation, Upf1 clamps onto the RNA, enclosing it in a channel formed by both the catalytic and regulatory domains. Upon binding to Upf2, the regulatory CH domain of Upf1 undergoes a large conformational change, causing the catalytic helicase domain to bind RNA less extensively and triggering its helicase activity. Formation of the surveillance complex thus modifies the RNA binding properties and the catalytic activity of Upf1, causing it to switch from an RNA-clamping mode to an RNA-unwinding mode.

Caught at its own game: regulatory small RNA inactivated by an inducible transcript mimicking its target.

A relevant, yet little recognized feature of antisense regulation is the possibility of switching roles between regulatory and regulated RNAs. Here we show that induction of a Salmonella gene relies on the conversion of a small RNA from effector to regulatory target. The chiP gene (formerly ybfM), identified and characterized in the present study, encodes a conserved enterobacterial chitoporin required for uptake of chitin-derived oligosaccharides. In the absence of inducer, chiP is kept silent by the action of a constitutively made small RNA, ChiX (formerly SroB, RybC), which pairs with a sequence at the 5' end of chiP mRNA. Silencing is relieved in the presence of chitooligosaccharides due to the accumulation of an RNA that pairs with ChiX and promotes its degradation. Anti-ChiX RNA originates from an intercistronic region of the chb operon, which comprises genes for chitooligosaccharide metabolism and whose transcription is activated in the presence of these sugars. We present evidence suggesting that the chb RNA destabilizes ChiX sRNA by invading the stem of its transcription terminator hairpin. Overall, our findings blur the distinction between effector and target in sRNA regulation, raising the possibility that some of the currently defined targets could actually be inhibitors of sRNA function.

Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains.

The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1.

Is open tracheotomy performed by residents in otorhinolaryngology a safe procedure? A retrospective cohort study.

Surgical or percutaneous tracheotomy is one of the commonest operations in the ENT practice and one of the first procedures to be taught to residents. No study exists that demonstrates the safety of this surgical procedure performed by unexperienced surgeons. The purpose was to compare outcomes of tracheotomies performed by supervised residents and surgeons in terms of postoperative complications and mortality, and identify risk factors for the onset of complications. Retrospective cohort study. Otolaryngology-Head and Neck Surgery Department, University of Florence, Italy. We included all patients undergoing tracheotomy from July 2008 to January 2013 and compared tracheotomies performed by supervised residents or surgeons. During the study period, 304 patients were submitted to tracheotomy. Patients operated by surgeons had a significantly higher number of tracheal rings fracture (p = 0.05), subcutaneous emphysema (p = 0.003) and tracheostomy tube displacement (p = 0.003), while supervised residents had a higher number of tracheitis/pneumonia (p = 0.04) as early complications. Patients operated by supervised residents had a significantly higher number of tube obstructions as late complication (p = 0.04). Using multivariate model, risk factors for early postoperative complications were male sex (p = 0.04) and delayed time to substitution with cuffless tube (p = 0.01), while only a trend to statistical significance was observed for urgent tracheotomies concerning the risk for late postoperative complications (p = 0.08). The current practice where residents perform tracheotomies supervised by a surgeon should not be disheartened. Our study demonstrates that it is safe and does not lead to higher risk of complications nor negatively affects the quality of care.

Subtotal supracricoid laryngectomy: changing in indications, surgical techniques and use of new surgical devices.

PURPOSE: The aim of this study is to evaluate the evolution of supracricoid partial laryngectomy (SCPL) in indications, surgical techniques and outcomes through last decades. MATERIALS AND METHODS: A retrospective analysis of 146 patients affected by laryngeal cancer treated with SCPL was carried on. We defined: (1) group A, 100 patients treated by cold instruments between 1995 and 2004; (2) group B, 46 patients treated by harmonic scalpel between 2005 and 2010. Complications rate, and functional and oncological results were documented and a comparison between the two groups was made; histopathological analysis of surgical margins was evaluated and correlated with local incidence of recurrence. RESULTS: Significant differences in age mean-value (p=0.02), T classification (p=0.007), and in indication for more advanced-staged patients were found in group B (p=0.001). Surgical procedure was shorter in group B (p<0.001), with shorter swallowing recovery (p=0.003). Oncological outcomes did not report any significant differences. Group B showed a higher incidence of post- operative arytenoid edema (p=0.03) associated with a lower rate of pneumonia (p=0.038). Despite a higher rate of close or positive-margins found in group B no higher incidence of local-recurrence was reported (p=0.02) compared to group A. CONCLUSIONS: We documented changing in indications and surgical technique for SCPL because of the development of modern diagnostic techniques and the introduction of low-thermal injury device allowing a more challenging tumor excision as well as with a shorter swallowing recovery in our series.

Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

ISG20: an enigmatic antiviral RNase targeting multiple viruses.

Interferon-stimulated gene 20 kDa protein (ISG20) is a relatively understudied antiviral protein capable of inhibiting a broad spectrum of viruses. ISG20 exhibits strong RNase properties, and it belongs to the large family of DEDD exonucleases, present in both prokaryotes and eukaryotes. ISG20 was initially characterized as having strong RNase activity in vitro, suggesting that its inhibitory effects are mediated via direct degradation of viral RNAs. This mechanism of action has since been further elucidated and additional antiviral activities of ISG20 highlighted, including direct degradation of deaminated viral DNA and translational inhibition of viral RNA and nonself RNAs. This review focuses on the current understanding of the main molecular mechanisms of viral inhibition by ISG20 and discusses the latest developments on the features that govern specificity or resistance to its action.

Surgical palliation in poorly differentiated neuroendocrine carcinoma of the hypopharynx: Case report.

BACKGROUND: Primary neuroendocrine carcinomas (NECs) are very rare entities accounting for 0.49% of all malignancies. Within the head and neck, the most common sites are the larynx and paranasal sinuses, while the hypopharynx is seldom described. CASE: We present a patient with a poorly differentiated metastatic NEC of the hypopharynx treated palliatively with organ-preserving surgery and post-operative chemotherapy, and literature review for well-documented pure hypopharyngeal NECs. Our patient died of chest infection during chemotherapy, 4 months after surgery. CONCLUSION: Chemotherapy remains the mainstay of treatment in the presence of metastases with 2-year overall survival of 15.7%. Due to the aggressive nature of poorly differentiated metastatic NECs, surgical management is seldom considered. We report and advocate the successful palliative role of organ-preserving, minimally invasive trans-oral LASER micro-surgery and neck dissection to control loco-regional head and neck disease, safe-guarding better quality of home life, despite limited life expectancy for this condition.

The C-Terminal Domain of HIV-1 Integrase: A Swiss Army Knife for the Virus?

Retroviral integrase is a multimeric enzyme that catalyzes the integration of reverse-transcribed viral DNA into the cellular genome. Beyond integration, the Human immunodeficiency virus type 1 (HIV-1) integrase is also involved in many other steps of the viral life cycle, such as reverse transcription, nuclear import, virion morphogenesis and proviral transcription. All these additional functions seem to depend on the action of the integrase C-terminal domain (CTD) that works as a molecular hub, interacting with many different viral and cellular partners. In this review, we discuss structural issues concerning the CTD, with particular attention paid to its interaction with nucleic acids. We also provide a detailed map of post-translational modifications and interaction with molecular partners.

Using oxygen dose histograms to quantify voxelised ultra-high dose rate (FLASH) effects in multiple radiation modalities.

Purpose.To introduce a methodology to predict tissue sparing effects in pulsed ultra-high dose rate radiation exposures which could be included in a dose-effect prediction system or treatment planning system and to illustrate it by using three published experiments.Methods and materials.The proposed system formalises the variability of oxygen levels as an oxygen dose histogram (ODH), which provides an instantaneous oxygen level at a delivered dose. The histogram concept alleviates the need for a mechanistic approach. At each given oxygen level the oxygen fixation concept is used to calculate the change in DNA-damage induction compared to the fully hypoxic case. Using the ODH concept it is possible to estimate the effect even in the case of multiple pulses, partial oxygen depletion, and spatial oxygen depletion. The system is illustrated by applying it to the seminal results by Town (Nat. 1967) on cell cultures and the pre-clinical experiment on cognitive effects by Montay-Gruelet al(2017Radiother. Oncol.124365-9).Results.The proposed system predicts that a possible FLASH-effect depends on the initial oxygenation level in tissue, the total dose delivered, pulse length and pulse repetition rate. The magnitude of the FLASH-effect is the result of a redundant system, in that it will have the same specific value for a different combination of these dependencies. The cell culture data are well represented, while a correlation between the pre-clinical experiments and the calculated values is highly significant (p < 0.01).Conclusions. A system based only on oxygen related effects is able to quantify most of the effects currently observed in FLASH-radiation.

Treatment planning comparison in the PROTECT-trial randomising proton versus photon beam therapy in oesophageal cancer: Results from eight European centres.

PURPOSE: To compare dose distributions and robustness in treatment plans from eight European centres in preparation for the European randomized phase-III PROTECT-trial investigating the effect of proton therapy (PT) versus photon therapy (XT) for oesophageal cancer. MATERIALS AND METHODS: All centres optimized one PT and one XT nominal plan using delineated 4DCT scans for four patients receiving 50.4 Gy (RBE) in 28 fractions. Target volume receiving 95% of prescribed dose (V95%iCTVtotal) should be >99%. Robustness towards setup, range, and respiration was evaluated. The plans were recalculated on a surveillance 4DCT (sCT) acquired at fraction ten and robustness evaluation was performed to evaluate the effect of respiration and inter-fractional anatomical changes. RESULTS: All PT and XT plans complied with V95%iCTVtotal >99% for the nominal plan and V95%iCTVtotal >97% for all respiratory and robustness scenarios. Lung and heart dose varied considerably between centres for both modalities. The difference in mean lung dose and mean heart dose between each pair of XT and PT plans was in median [range] 4.8 Gy [1.1;7.6] and 8.4 Gy [1.9;24.5], respectively. Patients B and C showed large inter-fractional anatomical changes on sCT. For patient B, the minimum V95%iCTVtotal in the worst-case robustness scenario was 45% and 94% for XT and PT, respectively. For patient C, the minimum V95%iCTVtotal was 57% and 72% for XT and PT, respectively. Patient A and D showed minor inter-fractional changes and the minimum V95%iCTVtotal was >85%. CONCLUSION: Large variability in dose to the lungs and heart was observed for both modalities. Inter-fractional anatomical changes led to larger target dose deterioration for XT than PT plans.

Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica.

Prokaryotic regulatory small RNAs act by a conserved mechanism and yet display a stunning structural variability. In the present study, we used mutational analysis to dissect the functional anatomy of RybB, a σ(E)-dependent sRNA that regulates the synthesis of major porins in Escherichia coli and Salmonella. Mutations in the chromosomal rybB locus that altered the expression of an ompC-lac fusion were identified. Some of the mutations cluster within a seven-nucleotide segment at the 5' end of the sRNA and affect its ability to pair with a sequence 40 nucleotides upstream from ompC translation start site. Other mutations map near the 3' end of RybB, destabilizing the sRNA or altering its binding to Hfq. The 5' end of RybB is also involved in ompD regulation. In this case, the sRNA can choose between two mutually exclusive pairing sites within the translated portion of the mRNA. Some of the RybB 5' end mutations affect the choice between the two sites, resulting in regulatory responses that diverge from those observed in ompC. Further analysis of RybB target specificity identified chiP (ybfM), a gene encoding an inducible chitoporin, as an additional member of the RybB regulon. Altogether, our results indicate that an heptameric 'seed' sequence is sufficient to confer susceptibility to RybB regulation.

Incorporating oxygenation levels in analytical DNA-damage models-quantifying the oxygen fixation mechanism.

Purpose.To develop a framework to include oxygenation effects in radiation therapy treatment planning which is valid for all modalities, energy spectra and oxygen levels. The framework is based on predicting the difference in DNA-damage resulting from ionising radiation at variable oxygenation levels.Methods.Oxygen fixation is treated as a statistical process in a simplified model of complex and simple damage. We show that a linear transformation of the microscopic oxygen fixation process allows to extend this to all energies and modalities, resulting in a relatively simple rational polynomial expression. The model is expanded such that it can be applied for polyenergetic beams. The methodology is validated using Microdosimetric Monte Carlo Damage Simulation code (MCDS). This serves as a bootstrap to determine relevant parameters in the analytical expression, as MCDS is shown to be extensively verified with published empirical data. Double-strand break induction as calculated by this methodology is compared to published proton experiments. Finally, an example is worked out where the oxygen enhancement ratio (OER) is calculated at different positions in a clinically relevant spread out Bragg peak (SOBP) dose deposition in water. This dose deposition is obtained using a general Monte Carlo code (FLUKA) to determine dose deposition and locate fluence spectra.Results.For all modalities (electrons, protons), the damage categorised as complex could be parameterised to within 0.3% of the value calculated using microdosimetric Monte Carlo. The proton beam implementation showed some variation in OERs which differed slightly depending on where the assessment was made; before the SOBP, mid-SOBP or at the distal edge. Environment oxygenation was seen to be the more important variable.Conclusions.An analytic expression calculating complex damage depending on modality, energy spectrum, and oxygenation levels was shown to be effective and can be readily incorporated in treatment planning software, to take into account the impact of variable oxygenation, forming a first step to an optimised treatment based on biological factors.

Assessment of robustness against setup uncertainties using probabilistic scenarios in lung cancer: a comparison of proton with photon therapy.

OBJECTIVE: We compared the sensitivity of intensity modulated proton therapy (IMPT) and photon volumetric modulated arc therapy (VMAT) plans to setup uncertainties in locally advanced non-small cell lung cancer (NSCLC) using probabilistic scenarios. METHODS: Minimax robust (MM) and planning target volume (PTV) optimised IMPT and VMAT nominal plans were created with physical dose of 70 Gy in 35 fractions in 10 representative patients. Using population data of setup errors, a fractionated treatment course was simulated, summed (Dsum) and compared to the nominal plan. Three treatment-course simulations were done for each plan. Target robustness criteria were: dose deviation of ≤5% to clinical target volume (CTV) D98% and CTV V95% ≥ 99.9%. Voxelwise simulation repeatability was analysed using Bland-Altman plots. Acceptable limits of agreement were 2% of the prescription dose. RESULTS: All Dsum met target robustness criteria. While fraction VMAT and MM-IMPT doses were excellent, simulated fraction doses in PTV-IMPT were suboptimal. Almost all (>99%) of VMAT and MM-IMPT fraction doses met both target robustness criteria. For PTV-IMPT, only 96.9 and 80.3% of fractions met CTVD98% and V95% criteria respectively. Simulation repeatability was excellent (limits of agreement range: 0.41-1.1 Gy) with strong positive correlations. CONCLUSION: When considering the whole treatment course, setup errors do not influence robustness irrespective of planning techniques used. However, on a fraction level, VMAT and MM-IMPT plans are superior compared to PTV-IMPT plans. ADVANCES IN KNOWLEDGE: Probabilistic analysis provides a fast and practical method for evaluating VMAT and IMPT plan sensitivity against setup uncertainty. VMAT and robust-optimised IMPT plans have comparable sensitivity to setup uncertainties in conventionally fractionated treatment for NSCLC.

Is an analytical dose engine sufficient for intensity modulated proton therapy in lung cancer?

OBJECTIVE: To identify a subgroup of lung cancer plans where the analytical dose calculation (ADC) algorithm may be clinically acceptable compared to Monte Carlo (MC) dose calculation in intensity modulated proton therapy (IMPT). METHODS: Robust-optimised IMPT plans were generated for 20 patients to a dose of 70 Gy (relative biological effectiveness) in 35 fractions in Raystation. For each case, four plans were generated: three with ADC optimisation using the pencil beam (PB) algorithm followed by a final dose calculation with the following algorithms: PB (PB-PB), MC (PB-MC) and MC normalised to prescription dose (PB-MC scaled). A fourth plan was generated where MC optimisation and final dose calculation was performed (MC-MC). Dose comparison and γ analysis (PB-PB vs PB-MC) at two dose thresholds were performed: 20% (D20) and 99% (D99) with PB-PB plans as reference. RESULTS: Overestimation of the dose to 99% and mean dose of the clinical target volume was observed in all PB-MC compared to PB-PB plans (median: 3.7 Gy(RBE) (5%) (range: 2.3 to 6.9 Gy(RBE)) and 1.8 Gy(RBE) (3%) (0.5 to 4.6 Gy(RBE))). PB-MC scaled plans resulted in significantly higher CTVD2 compared to PB-PB (median difference: -4 Gy(RBE) (-6%) (-5.3 to -2.4 Gy(RBE)), p ≤ .001). The overall median γ pass rates (3%-3 mm) at D20 and D99 were 93.2% (range:62.2-97.5%) and 71.3 (15.4-92.0%). On multivariate analysis, presence of mediastinal disease and absence of range shifters were significantly associated with high γ pass rates. Median D20 and D99 pass rates with these predictors were 96.0% (95.3-97.5%) and 85.4% (75.1-92.0%). MC-MC achieved similar target coverage and doses to OAR compared to PB-PB plans. CONCLUSION: In the presence of mediastinal involvement and absence of range shifters Raystation ADC may be clinically acceptable in lung IMPT. Otherwise, MC algorithm would be recommended to ensure accuracy of treatment plans. ADVANCES IN KNOWLEDGE: Although MC algorithm is more accurate compared to ADC in lung IMPT, ADC may be clinically acceptable where there is mediastinal involvement and absence of range shifters.

In vitro, in cellulo and structural characterizations of the interaction between the integrase of Porcine Endogenous Retrovirus A/C and proteins of the BET family.

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.