Protein interactions during coronavirus assembly.

Coronaviruses assemble and obtain their envelope at membranes of the intermediate compartment between the endoplasmic reticulum and Golgi complex. Like other enveloped viruses, coronavirus assembly is presumably dependent on protein localization and protein-protein as well as protein-RNA interactions. We have used the bovine coronavirus (BCV) as a model to study interactions between the viral proteins in virus-infected cells that are important for coronavirus assembly. BCV is a prototype for the coronaviruses that express an additional major structural protein, the hemagglutinin esterase (HE), in addition to the spike (S) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. Complexes consisting of the M, S, and HE proteins were detected in virus-infected cells by coimmunoprecipitations. Kinetic analyses demonstrated that S protein and HE each quickly formed a complex with M protein after synthesis, whereas heterocomplexes consisting of all three proteins formed more slowly. The kinetics of HE biosynthesis revealed that the half-life of oligomerization was approximately 30 min, which correlated with the appearance of complexes consisting of M, HE, and S proteins, suggesting that oligomerization and/or conformational changes may be important for the S-M-HE protein complexes to form. Only HE dimers were found associated with the heterocomplexes consisting of all three proteins. S-M-HE protein complexes were detected prior to processing of the oligosaccharide chains on HE, indicating that these protein complexes formed in a premedial Golgi compartment before trimming of sugar chains. Transient coexpressions and double-labeling immunofluorescence demonstrated that HE and S proteins colocalized with M protein. This was further supported by coimmunoprecipitation of specific HE-M and S-M protein complexes from transfected cells, indicating that these proteins can form complexes in the absence of other viral proteins.     

Etude comparee des proprietes physico-chimiques des mannanes de neuf levures

In order to correlate and compare the antigenic relationships observed between nine yeast strains, we have studied their major antigenic determinant represented by the mannans. This material is a constituent of their cell.The compounds that we have isolated are, in reality, glycopeptides. All their polysaccharide fractions consist of mannose only — except for those of the compounds that we have isolated of R. glutinis and C. lipolytica. All these polysaccharides have very close ramified structures; the only one that seems to be very different is that of R. glutinis, and this is in agreement with the antigenic properties of that yeast.The peptidic fractions of these glycopeptides are rich in threonine, serine and aspartic acid and it is very likely that these amino acids make the link with the polysaccharide fraction. The quantity of these amino acids seem to be affected by the conditions of culture of the yeasts, but not their presence or absence.     

CBEA: Competitive balances for taxonomic enrichment analysis

Research in human-associated microbiomes often involves the analysis of taxonomic count tables generated via high-throughput sequencing. It is difficult to apply statistical tools as the data is high-dimensional, sparse, and compositional. An approachable way to alleviate high-dimensionality and sparsity is to aggregate variables into pre-defined sets. Set-based analysis is ubiquitous in the genomics literature and has demonstrable impact on improving interpretability and power of downstream analysis. Unfortunately, there is a lack of sophisticated set-based analysis methods specific to microbiome taxonomic data, where current practice often employs abundance summation as a technique for aggregation. This approach prevents comparison across sets of different sizes, does not preserve inter-sample distances, and amplifies protocol bias. Here, we attempt to fill this gap with a new single-sample taxon enrichment method that uses a novel log-ratio formulation based on the competitive null hypothesis commonly used in the enrichment analysis literature. Our approach, titled competitive balances for taxonomic enrichment analysis (CBEA), generates sample-specific enrichment scores as the scaled log-ratio of the subcomposition defined by taxa within a set and the subcomposition defined by its complement. We provide sample-level significance testing by estimating an empirical null distribution of our test statistic with valid p-values. Herein, we demonstrate, using both real data applications and simulations, that CBEA controls for type I error, even under high sparsity and high inter-taxa correlation scenarios. Additionally, CBEA provides informative scores that can be inputs to downstream analyses such as prediction tasks.     

AvrRps4 effector family processing and recognition in lettuce

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector‐triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N‐terminus (AvrRps4^N) and the C‐terminus (AvrRps4^C). AvrRps4^C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4‐mediated immunity in Arabidopsis; on the other hand, AvrRps4^N induces HR in lettuce. Furthermore, AvrRps4^N‐mediated HR requires a conserved arginine at position 112 (R112), which is also important for full‐length AvrRps4 (AvrRps4^F) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO‐mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.