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

Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation

The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models

Nutrient limitations to bacterial and fungal growth during cellulose decomposition in tropical forest soils

Nutrients constrain the soil carbon cycle in tropical forests, but we lack knowledge on how these constraints vary within the soil microbial community. Here, we used in situ fertilization in a montane tropical forest and in two lowland tropical forests on contrasting soil types to test the principal hypothesis that there are different nutrient constraints to different groups of microorganisms during the decomposition of cellulose. We also tested the hypotheses that decomposers shift from nitrogen to phosphorus constraints from montane to lowland forests, respectively, and are further constrained by potassium and sodium deficiency in the western Amazon. Cellulose and nutrients (nitrogen, phosphorus, potassium, sodium, and combined) were added to soils in situ, and microbial growth on cellulose (phospholipid fatty acids and ergosterol) and respiration were measured. Microbial growth on cellulose after single nutrient additions was highest following nitrogen addition for fungi, suggesting nitrogen as the primary limiting nutrient for cellulose decomposition. This was observed at all sites, with no clear shift in nutrient constraints to decomposition between lowland and montane sites. We also observed positive respiration and fungal growth responses to sodium and potassium addition at one of the lowland sites. However, when phosphorus was added, and especially when added in combination with other nutrients, bacterial growth was highest, suggesting that bacteria out-compete fungi for nitrogen where phosphorus is abundant. In summary, nitrogen constrains fungal growth and cellulose decomposition in both lowland and montane tropical forest soils, but additional nutrients may also be of critical importance in determining the balance between fungal and bacterial decomposition of cellulose

Training future generations to deliver evidence-based conservation and ecosystem management

Data availability statement: No data was used in this study.Peer review: The peer review history for this article is available at: https://publons.com/publon/10.1002/2688-8319.12032.Supporting Information: eso312032-sup-0001-SuppMat.docx (21.1 KB) available at: https://besjournals.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1002%2F2688-8319.12032&file=eso312032-sup-0001-SuppMat.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.Copyright © 2021 The Authors. 1. To be effective, the next generation of conservation practitioners and managers need to be critical thinkers with a deep understanding of how to make evidence-based decisions and of the value of evidence synthesis. 2. If, as educators, we do not make these priorities a core part of what we teach, we are failing to prepare our students to make an effective contribution to conservation practice. 3. To help overcome this problem we have created open access online teaching materials in multiple languages that are stored in Applied Ecology Resources. So far, 117 educators from 23 countries have acknowledged the importance of this and are already teaching or about to teach skills in appraising or using evidence in conservation decision-making. This includes 145 undergraduate, postgraduate or professional development courses. 4. We call for wider teaching of the tools and skills that facilitate evidence-based conservation and also suggest that providing online teaching materials in multiple languages could be beneficial for improving global understanding of other subject areas. Making informed conservation and ecosystem management choices is based upon a sound understanding of the relevant evidence. There is an increasing wealth of conservation science available, and access to this is becoming easier. But, are conservation practitioners being trained to utilize this information? In conservation, decision-making is often based upon past experience or expert knowledge, as opposed to the full body of scientific literature (e.g., Pullin, Knight, Stone, & Charman, 2004; Rafidimanantsoa, Poudyal, Ramamonjisoa, & Jones, 2018). The failure to include scientific evidence in decision-making has the potential to reduce the effectiveness of management, or even lead to detrimental actions being undertaken (Walsh, Dicks, & Sutherland, 2015). Evidence-based conservation (EBC) seeks to avoid this by providing tools to facilitate and inform decision-making. To do this, scientific evidence is collated and critically appraised for its quality and relevance, and integrated with other knowledge, experience, values and costs (Sutherland, Pullin, Dolman, & Knight, 2004). Wider adoption of EBC requires conservation professionals to be trained in its principles and taught how to use it to inform conservation decision-making.MAVA Foundation; Arcadia Fund

Comparison of antemortem antimicrobial treatment regimens to antimicrobial susceptibility patterns of postmortem lung isolates from feedlot cattle with bronchopneumonia

A retrospective study was performed to compare the treatment regimens in feedlot cattle that died with bovine respiratory disease (BRD) to the antimicrobial susceptibility patterns of the microorganisms isolated from lungs. Forty-three cattle submitted by the Willard Sparks Beef Research Center (WSBRC) to the Oklahoma Animal Disease Diagnostic Laboratory for postmortem examination during 2007 had bronchopneumonia (acute = 16, subacute = 5, or chronic = 22). Lungs from cattle were cultured aerobically (40 cattle) and for Mycoplasma spp. (34 cattle). Susceptibility panels were performed. At least 1 BRD pathogen (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis, or Arcanobacterium pyogenes) was isolated from 39 cattle, and 77% (30/39) had multiple organisms recovered. Mycoplasmal infections were common (25/34) and a major component of mixed infections (24/25). The majority (60%) of the M. haemolytica, P. multocida, and H. somni isolates were resistant to tetracycline. Most of the H. somni isolates (67%) were susceptible to tilmicosin (Ti), enrofloxacin (En), ceftiofur (Ce), and florfenicol, despite extensive treatment with Ti, En, and Ce (75% of isolates were from cattle that received each antimicrobial once). Most of the M. haemolytica (65%) and P. multocida (79%) isolates were susceptible to En and Ce, despite antemortem treatment of cattle with these antimicrobials. Hence, the current study reports a discrepancy between the antemortem treatment of clinical BRD and the susceptibility patterns of the bacteria isolated from lungs postmortem. Based on these findings, factors other than antimicrobial resistance are playing a role in the death of feedlot cattle with BRD