Inhibition of Henipavirus fusion and infection by heptad-derived peptides of the Nipah virus fusion glycoprotein

Background

Oral infection of infant macaques with simian immunodeficiency virus (SIV) is a useful animal model to test interventions to reduce postnatal HIV transmission via breast-feeding. We previously demonstrated that immunization of infant rhesus macaques with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol and Env, or live-attenuated SIVmac1A11 resulted in lower viremia and longer survival compared to unimmunized controls after oral challenge with virulent SIVmac251 (Van Rompay et al., J. Virology 77:179–190, 2003). Here we evaluate the impact of these vaccines on oral transmission and evolution of SIV envelope variants.

Results

Limiting dilution analysis of SIV RNA followed by heteroduplex mobility assays of the V1–V2 envelope (env) region revealed two major env variants in the uncloned SIVmac251 inoculum. Plasma sampled from all infants 1 week after challenge contained heterogeneous SIV env populations including one or both of the most common env variants in the virus inoculum; no consistent differences in patterns of env variants were found between vaccinated and unvaccinated infants. However, SIV env variant populations diverged in most vaccinated monkeys 3 to 5 months after challenge, in association with the development of neutralizing antibodies.

Conclusions

These patterns of viral envelope diversity, immune responses and disease course in SIV-infected infant macaques are similar to observations in HIV-infected children, and underscore the relevance of this pediatric animal model. The results also support the concept that neonatal immunization with HIV vaccines might modulate disease progression in infants infected with HIV by breast-feeding.     

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization,phase separation,and RNA splicing

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.     

The Mechanism of Henipavirus Fusion： Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.     

Bias and Sampling Error of the Estimated Proportion of Genotypic Variance Explained by Quantitative Trait Loci Determined From Experimental Data in Maize Using Cross Validation and Validation With Independent Samples.

Cross validation (CV) was used to analyze the effects of different environments and different genotypic samples on estimates of the proportion of genotypic variance explained by QTL (p). Testcrosses of 344 F(3) maize lines grown in four environments were evaluated for a number of agronomic traits. In each of 200 replicated CV runs, this data set was subdivided into an estimation set (ES) and various test sets (TS). ES were used to map QTL and estimate p for each run (p(ES)) and its median (p(ES)) across all runs. The bias of these estimates was assessed by comparison with the median (p(TS.ES)) obtained from TS. We also used two independent validation samples derived from the same cross for further comparison. The median p(ES) showed a large upward bias compared to p(TS.ES). Environmental sampling generally had a smaller effect on the bias of p(ES) than genotypic sampling or both factors simultaneously. In independent validation, p(TS.ES) was on average only 50% of p(ES). A wide range among p(ES) reflected a large sampling error of these estimates. QTL frequency distributions and comparison of estimated QTL effects indicated a low precision of QTL localization and an upward bias in the absolute values of estimated QTL effects from ES. CV with data from three QTL studies reported in the literature yielded similar results as those obtained with maize testcrosses. We therefore recommend CV for obtaining asymptotically unbiased estimates of p and consequently a realistic assessment of the prospects of MAS.     

Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production

In analyzing the reductive power of Escherichia coli K-12 for metabolic engineering approaches, we identified YahK and YjgB, two medium-chain dehydrogenases/reductases subgrouped to the cinnamyl alcohol dehydrogenase family, as being important. Identification was achieved using a stepwise purification protocol starting with crude extract. For exact characterization, the genes were cloned into pET28a vector and expressed with N-terminal His tag. Substrate specificity studies revealed that a large variety of aldehydes but no ketones are converted by both enzymes. YahK and and YjgB strongly preferred NADPH as cofactor. The structure of YjgB was modeled using YahK as template for a comparison of the active center giving a first insight to the different substrate preferences. The enzyme activity for YahK, YjgB, and YqhD was determined on the basis of the temperature. YahK showed a constant increase in activity until 60 °C, whereas YjgB was most active between 37 and 50 °C. YqhD achieved the highest activity at 50 °C. Comparing YjgB and Yahk referring to the catalytic efficiency, YjgB achieved for almost all substrates higher rates (butyraldehyde 221 s^?1?mM^?1, benzaldehyde 1,305 s^?1?mM^?1). Exceptions are the two substrates glyceraldehydes (no activity for YjgB) and isobutyraldehyde (YjgB 0.26 s^?1?mM^?1) which are more efficiently converted by YahK (glyceraldehyde 2.8 s^?1?mM^?1, isobutyraldehyde 14.6 s^?1?mM^?1). YahK and even more so YjgB are good candidates for the reduction of aldehydes in metabolic engineering approaches and could replace the currently used YqhD.     

Remote sensing as a data source to analyse regional implications of genetically modified plants in agriculture—Oilseed rape (Brassica napus) in Northern Germany

Remote sensing permits the identification of locations and area extent where particular crops are cultivated across larger regions. This requires the availability of crop- and region-specific algorithms. We developed an approach to identify oilseed rape fields in Northern Germany. The remote sensing data sources and main processing steps are described. The derived cultivation density information provides an overview of the fine-scale spatial structure and crop neighbourhood relations in Northern Germany as one of the main oilseed rape cultivation regions in Europe. Geographical Information System (GIS) analyses involving buffer operations allowed to identify those parts of the region, where the highest interaction potential of GM crops and conventional crops would occur if GM varieties were admitted for cultivation. Cultivation density and field sizes in combination were used to indicate the interaction intensity as a marker for observation requirement (environmental monitoring) and for those potential risks that relate to density parameter. Conclusions can be drawn for the feasibility of coexistence measures when neighbouring farmers have to co-operate to keep separation distances between GM crop cultivation and conventional varieties. Together with other data sources, the results of satellite image analysis can be used as input data for up-scaling small-scale model results. Remote sensing data allow to specify, which field density parameter must be chosen to cover the regional variability of cultivation conditions, in particular the regional distribution of field sizes and field density.     

Forespore engulfment mediated by a ratchet-like mechanism

A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.