Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter.

Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 3' and 5' vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 5' end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.

Capping pores of alphavirus nsP1 gate membranous viral replication factories.

Positive-sense single-stranded RNA viruses, such as coronaviruses, flaviviruses and alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells1-7. The remodelling of the host-cell membranes for the formation of these organelles is coupled to the membrane association of viral replication complexes and to RNA synthesis. These viral niches allow for the concentration of metabolites and proteins for the synthesis of viral RNA, and prevent the detection of this RNA by the cellular innate immune system8. Here we present the cryo-electron microscopy structure of non-structural protein 1 (nsP1) of the alphavirus chikungunya virus, which is responsible for RNA capping and membrane binding of the viral replication machinery. The structure shows the enzyme in its active form, assembled in a monotopic membrane-associated dodecameric ring. The structure reveals the structural basis of the coupling between membrane binding, oligomerization and allosteric activation of the capping enzyme. The stoichiometry-with 12 active sites in a single complex-redefines viral replication complexes as RNA synthesis reactors. The ring shape of the complex implies it has a role in controlling access to the viral organelle and ensuring the exit of properly capped viral RNA. Our results provide high-resolution information about the membrane association of the replication machinery of positive-sense single-stranded RNA viruses, and open up avenues for the further characterization of viral replication on cell membranes and the generation of antiviral agents.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores.

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Serum mass spectrometry for treatment monitoring in patients with multiple myeloma receiving ARI0002h CAR T-cells.

Chimeric antigen receptor (CAR) T-cell therapies have increased the patients with relapsed/refractory multiple myeloma (RRMM) in whom standard electrophoretic techniques fail to detect the M-protein. Quantitative immunoprecipitation mass spectrometry (QIP-MS) can accurately measure serum M-protein with high sensitivity, and identify interferences caused by therapeutic monoclonal antibodies. Here, we investigate the outcome of QIP-MS in 33 patients treated with the academic BCMA-directed CAR T-cell ARI0002h (Cesnicabtagene Autoleucel). QIP-MS offered more detailed insights than serum immunofixation (sIFE), identifying glycosylated M-proteins and minor additional peaks. Moreover, the potential interferences owing to daratumumab or tocilizumab treatments were successfully detected. When analysing different assay platforms during patient's monitoring after ARI0002h administration, we observed that QIP-MS showed a high global concordance (78.8%) with sIFE, whereas it was only moderate (55.6%) with bone marrow (BM)-based next-generation flow cytometry (NGF). Furthermore, QIP-MS consistently demonstrated the lowest negativity rate across the different timepoints (27.3% vs. 60.0% in months 1 and 12, respectively). Patients with QIP-MS(+)/BM-based NGF(-) showed a non-significant shorter median progression free survival than those with QIP-MS(-)/BM-based NGF(-). In summary, we show the first experience to our knowledge demonstrating that QIP-MS could be particularly useful as a non-invasive technique when evaluating response after CAR T-cell treatment in MM.

Follow-up of Immune Response in Patients with Common Variable Immunodeficiency following SARS-CoV-2 Vaccination.

The COVID-19 pandemic highlighted the importance of effective vaccination strategies in controlling the spread of infectious diseases. SARS-CoV-2 vaccine has demonstrated high efficacy in preventing COVID-19 infection in the general population. However, the efficacy of this vaccine in patients with predominantly antibody deficiencies, such as common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA), should be closely monitored. CVID and XLA are rare genetic disorders that impair the immune system's ability to produce antibodies, which are crucial for fighting infections. Patients with these disorders have a higher risk of severe disease and mortality from COVID-19 due to their compromised immune systems. In this study, we evaluated the humoral and cellular immune responses after four doses of mRNA-1273 and one BNT162b2 bivalent vaccine in a cohort of patients with CVID and XLA. The response in this population was lower than in the control group. However, the administration of the third dose improved the number of patients with seroconversion and the intensity of the humoral response, as well as the number of patients with a positive cellular response. Finally, the administration of the fourth and fifth doses improves the antibody titer and neutralization against wild type variant, but not against the prevalent XBB1.5 variant.

Fractionated initial infusion and booster dose of ARI0002h, a humanised, BCMA-directed CAR T-cell therapy, for patients with relapsed or refractory multiple myeloma (CARTBCMA-HCB-01): a single-arm, multicentre, academic pilot study.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is a promising option for patients with heavily treated multiple myeloma. Point-of-care manufacturing can increase the availability of these treatments worldwide. We aimed to assess the safety and activity of ARI0002h, a BCMA-targeted CAR T-cell therapy developed by academia, in patients with relapsed or refractory multiple myeloma. METHODS: CARTBCMA-HCB-01 is a single-arm, multicentre study done in five academic centres in Spain. Eligible patients had relapsed or refractory multiple myeloma and were aged 18-75 years; with an Eastern Cooperative Oncology Group performance status of 0-2; two or more previous lines of therapy including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody; refractoriness to the last line of therapy; and measurable disease according to the International Myeloma Working Group criteria. Patients received an initial fractionated infusion of 3 × 106 CAR T cells per kg bodyweight in three aliquots (0·3, 0·9, and 1·8 × 106 CAR-positive cells per kg intravenously on days 0, 3, and 7) and a non-fractionated booster dose of up to 3 × 106 CAR T cells per kg bodyweight, at least 100 days after the first infusion. The primary endpoints were overall response rate 100 days after first infusion and the proportion of patients developing cytokine-release syndrome or neurotoxic events in the first 30 days after receiving treatment. Here, we present an interim analysis of the ongoing trial; enrolment has ended. This study is registered with ClinicalTrials.gov, NCT04309981, and EudraCT, 2019-001472-11. FINDINGS: Between June 2, 2020, and Feb 24, 2021, 44 patients were assessed for eligibility, of whom 35 (80%) were enrolled. 30 (86%) of 35 patients received ARI0002h (median age 61 years [IQR 53-65], 12 [40%] were female, and 18 [60%] were male). At the planned interim analysis (cutoff date Oct 20, 2021), with a median follow-up of 12·1 months (IQR 9·1-13·5), overall response during the first 100 days from infusion was 100%, including 24 (80%) of 30 patients with a very good partial response or better (15 [50%] with complete response, nine [30%] with very good partial response, and six [20%] with partial response). Cytokine-release syndrome was observed in 24 (80%) of 30 patients (all grade 1-2). No cases of neurotoxic events were observed. Persistent grade 3-4 cytopenias were observed in 20 (67%) patients. Infections were reported in 20 (67%) patients. Three patients died: one because of progression, one because of a head injury, and one due to COVID-19. INTERPRETATION: ARI0002h administered in a fractioned manner with a booster dose after 3 months can provide deep and sustained responses in patients with relapsed or refractory multiple myeloma, with a low toxicity, especially in terms of neurological events, and with the possibility of a point-of-care approach. FUNDING: Instituto de Salud Carlos III (co-funded by the EU), Fundación La Caixa, and Fundació Bosch i Aymerich.

Axicabtagene ciloleucel compared to tisagenlecleucel for the treatment of aggressive B-cell lymphoma.

Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) are CD19-targeted chimeric antigen receptor (CAR) T cells approved for relapsed/refractory (R/R) large B-cell lymphoma (LBCL). We performed a retrospective study to evaluate safety and efficacy of axi-cel and tisa-cel outside the setting of a clinical trial. Data from consecutive patients with R/R LBCL who underwent apheresis for axi-cel or tisa-cel were retrospectively collected from 12 Spanish centers. A total of 307 patients underwent apheresis for axi-cel (n=152) and tisa-cel (n=155) from November 2018 to August 2021, of which 261 (85%) received a CAR T infusion (88% and 82%, respectively). Median time from apheresis to infusion was 41 days for axi-cel and 52 days for tisa-cel (P=0.006). None of the baseline characteristics were significantly different between both cohorts. Both cytokine release syndrome and neurologic events (NE) were more frequent in the axi-cel group (88% vs. 73%, P=0.003, and 42% vs. 16%, P<0.001, respectively). Infections in the first 6 months post-infusion were also more common in patients treated with axi-cel (38% vs. 25%, P=0.033). Non-relapse mortality was not significantly different between the axi-cel and tisa-cel groups (7% and 4%, respectively, P=0.298). With a median follow-up of 9.2 months, median PFS and OS were 5.9 and 3 months, and 13.9 and 11.2 months for axi-cel and tisa-cel, respectively. The 12-month PFS and OS for axi-cel and tisa-cel were 41% and 33% (P=0.195), 51% and 47% (P=0.191), respectively. Factors associated with lower OS in the multivariate analysis were increased lactate dehydrogenase, ECOG ≥2 and progressive disease before lymphodepletion. Safety and efficacy results in our real-world experience were comparable with those reported in the pivotal trials. Patients treated with axi-cel experienced more toxicity but similar non-relapse mortality compared with those receiving tisa-cel. Efficacy was not significantly different between both products.

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro.

Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.

Structural Insights into the Mechanisms of Action of Functionally Distinct Classes of Chikungunya Virus Nonstructural Protein 1 Inhibitors.

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-ß-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.

Structure and function of the Toscana virus cap-snatching endonuclease.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

Predicting Survival after Allogeneic Hematopoietic Cell Transplantation in Myelofibrosis: Performance of the Myelofibrosis Transplant Scoring System (MTSS) and Development of a New Prognostic Model.

Accurate prognostic tools are crucial to assess the risk/benefit ratio of allogeneic hematopoietic cell transplantation (allo-HCT) in patients with myelofibrosis (MF). We aimed to evaluate the performance of the Myelofibrosis Transplant Scoring System (MTSS) and identify risk factors for survival in a multicenter series of 197 patients with MF undergoing allo-HCT. After a median follow-up of 3.1 years, 47% of patients had died, and the estimated 5-year survival rate was 51%. Projected 5-year risk of nonrelapse mortality and relapse incidence was 30% and 20%, respectively. Factors independently associated with increased mortality were a hematopoietic cell transplantation-specific comorbidity index (HCT-CI) ≥3 and receiving a graft from an HLA-mismatched unrelated donor or cord blood, whereas post-transplant cyclophosphamide (PT-Cy) was associated with improved survival. Donor type was the only parameter included in the MTSS model with independent prognostic value for survival. According to the MTSS, 3-year survival was 62%, 66%, 37%, and 17% for low-, intermediate-, high-, and very high-risk groups, respectively. By pooling together the low- and intermediate-risk groups, as well as the high- and very high-risk groups, we pinpointed 2 categories: standard risk and high risk (25% of the series). Three-year survival was 62% in standard-risk and 25% in high-risk categories (P < .001). We derived a risk score based on the 3 independent risk factors for survival in our series (donor type, HCT-CI, and PT-Cy). The corresponding 5-year survival for the low-, intermediate-, and high-risk categories was 79%, 55%, and 32%, respectively (P < .001). In conclusion, the MTSS model failed to clearly delineate 4 prognostic groups in our series but may still be useful to identify a subset of patients with poor outcome. We provide a simple prognostic scoring system for risk/benefit considerations before transplantation in patients with MF.

Towards the elimination of hepatitis C: implementation of reflex testing in Andalusia.

BACKGROUND AND AIM: undiagnosed hepatitis C virus (HCV) infection and/or inadequate access to care are barriers to the elimination of HCV. Reflex testing has proven to facilitate referral to care, treatment and viral elimination. In this study, a reflex testing program was implemented in Andalusia and its impact on access to care was evaluated. PATIENTS AND METHODS: an observational, retrospective and prospective study was performed across diagnostic laboratories responsible for HCV diagnosis in southern Spain. After surveying the barriers to performing reflex testing, the number of patients that were not referred for care in 2016 was retrospectively studied (pre-reflex cohort). Subsequently, several measures were proposed to overcome the identified barriers. Finally, reflex testing was implemented and its impact evaluated. RESULTS: the pre-reflex cohort included information from 1,053 patients. Slightly more than half of the patients (n = 580; 55%) visited a specialist for treatment evaluation during a median period of 71 days (interquartile range = 35-134) since the date of diagnosis. The post-reflex cohort (September 2017 to March 2018) included 623 patients. Only 17% (n = 106) of the patients had not been referred for care or evaluated for treatment in a median period of 52 days (interquartile range = 28-86). CONCLUSIONS: in 2016, nearly half of new HCV diagnoses in southern Spain were not referred for care. Barriers to the implementation of reflex testing were overcome in our study. Moreover, this strategy was effectively implemented in 2017. Reflex testing contributed to improving referral for care. This program will contribute to the micro-elimination of hepatitis C in Spain.

Structural insights into reptarenavirus cap-snatching machinery.

Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.

Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases.

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5' end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase.

Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein.

The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged N- and C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.

Systems to establish bunyavirus genome replication in the absence of transcription.

The L polymerase of bunyaviruses replicates and transcribes the viral genome. While replication products are faithful copies of the uncapped genomic RNA, transcription products contain capped 5' extensions which had been cleaved from host cell mRNAs. For La Crosse virus (LACV; genus Orthobunyavirus), the nuclease responsible for host cell mRNA cleavage is located at the N terminus of the L protein, with an active site of five conserved amino acids (H34, D52, D79, D92, and K94) surrounding two Mn(2+) ions (J. Reguera, F. Weber, and S. Cusack, PLoS Pathog. 6:e1001101, 2010). Here, we present reverse genetics systems and L mutants enabling us to study bunyaviral genome replication in the absence of transcription. Transcription was evaluated with an enhanced minigenome system consisting of the viral polymerase L, nucleocapsid protein N, a negative-sense minigenome, and--to alleviate antiviral host responses--a dominant-negative mutant (PKRΔE7) of the antiviral kinase protein kinase R (PKR). The transcriptional activity was strongly reduced by mutation of any of the five key amino acids, and the H34K, D79A, D92A, and K94A LACV L mutants were almost entirely silent in transcription. The replication activity of the L mutants was measured by packaging of progeny minigenomes into virus-like particles (VLPs). All mutant L proteins except K94A retained full replication activity. To test the broader applicability of our results, we introduced the homolog of mutation D79A (D111A) into the L sequence of Rift Valley fever virus (RVFV; genus Phlebovirus). As for LACV D79A, the RVFV D111A was incapable of transcription but fully active in replication. Thus, we generated mutants of LACV and RVFV L polymerases that are specifically deficient in transcription. Genome replication by bunyavirus polymerases can now be studied in the absence of transcription using convenient reverse genetics systems.

Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

The coronaviruses (CoVs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN), a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S) mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs) of two closely related CoV strains, transmissible gastroenteritis virus (TGEV) and porcine respiratory CoV (PRCV), in complex with their receptor, porcine APN (pAPN), or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

Biophysical and structural study of La Crosse virus endonuclease inhibition for the development of new antiviral options.

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.

Evaluation of a new CT/NG/TV/MG Real-Time PCR Kit (Vircell) versus the Allplex STI Essential Assay (Seegene) for the diagnosis of sexually transmitted infections.

Sexually transmitted infections (STI) are a public health problem. Real-time PCR assays are the most sensitive test for screening and diagnosis of these infections. The aim of this study was to evaluate a new CT/NG/TV/MG Real-Time PCR (RT-PCR) kit (Vircell) for the detection of Chamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis for the diagnosis of sexual transmitted infections using the Allplex STI Essential Assay (Seegene) as the reference's method. A total of 497 samples from different anatomical sites (endocervical, urethral, rectal, pharyngeal and urine) were analysed from October 2022 to February 2023. A total of 108 (21.73 %) and 106 (21.33 %) positive samples were found for any of the assays used. The most commonly detected pathogen was N. gonorrhoeae (52 samples; 10.46 %), and the least commonly detected was T. vaginalis (three samples; 0.60 %). The anatomical site with the highest prevalence of micro-organisms was a non-urogenital site, the pharynx (26 positive samples; 5.23 %). Using the Allplex STI Essential Assay (Seegene) as the reference method, the diagnosis performance showed that the average specificity of CT/NG/TV/MG RT-PCR Kit (Vircell) was 99.84 % and the sensitivity was 99.53 %. The overall concordance was k=0.98 (CI95 %; 0.96-1). In conclusion, the CT/NG/TV/MG RT-PCR Kit (Vircell) assay shows a good sensitivity and specificity and constitutes a promising and additional alternative to routine procedures for distinct types of clinical specimen in diagnosis STI.