Genomic Characterization of a New Enamovirus Infecting Common Bean

A novel enamovirus was identified from bean plants with disease symptoms. Its genome of 5,781 nucleotides (nt) encodes five open reading frames. The virus and other species of the genus Enamovirus share identities of 50.4%-68.4% at the complete genome, and 19.9%-51.9% of P0, 24.9%-52.5% of P1, 33.4%-62.9% of P1-P2, 30.6%-81.1% of P3, 32.3%-74.2% of P3-P5 at amino acid sequence level, respectively. Phylogenetic analysis showed that the virus is most closely related to Alfalfa enamovirus 1 and Pea enation mosaic virus 1 in the genus Enamovirus within family Solemoviridae. These results suggest that the virus should be considered as a novel species in the genus Enamovirus and tentatively named as “bean enamovirus 1”.

Chapter 2b: The molecular antigenic structure of the TBEV

TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.

Resilin-mimetics as a smart biomaterial platform for biomedical applications

Intrinsically disordered proteins have dramatically changed the structure–function paradigm of proteins in the 21st century. Resilin is a native elastic insect protein, which features intrinsically disordered structure, unusual multi-stimuli responsiveness and outstanding resilience. Advances in computational techniques, polypeptide synthesis methods and modular protein engineering routines have led to the development of novel resilin-like polypeptides (RLPs) including modular RLPs, expanding their applications in tissue engineering, drug delivery, bioimaging, biosensors, catalysis and bioelectronics. However, how the responsive behaviour of RLPs is encoded in the amino acid sequence level remains elusive. This review summarises the milestones of RLPs, and discusses the development of modular RLP-based biomaterials, their current applications, challenges and future perspectives. A perspective of future research is that sequence and responsiveness profiling of RLPs can provide a new platform for the design and development of new modular RLP-based biomaterials with programmable structure, properties and functions.

Chapter 2b: The molecular antigenic structure of the TBEV

TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

Conserved regions in long non-coding RNAs contain abundant translation and protein–RNA interaction signatures

The mammalian transcriptome includes thousands of transcripts that do not correspond to annotated protein-coding genes and that are known as long non-coding RNAs (lncRNAs). A handful of lncRNAs have well-characterized regulatory functions but the biological significance of the majority of them is not well understood. LncRNAs that are conserved between mice and humans are likely to be enriched in functional sequences. Here, we investigate the presence of different types of ribosome profiling signatures in lncRNAs and how they relate to sequence conservation. We find that lncRNA-conserved regions contain three times more ORFs with translation evidence than non-conserved ones, and identify nine cases that display significant sequence constraints at the amino acid sequence level. The study also reveals that conserved regions in intergenic lncRNAs are significantly enriched in protein–RNA interaction signatures when compared to non-conserved ones; this includes sites in well-characterized lncRNAs, such as Cyrano, Malat1, Neat1 and Meg3, as well as in tens of lncRNAs of unknown function. This work illustrates how the analysis of ribosome profiling data coupled with evolutionary analysis provides new opportunities to explore the lncRNA functional landscape.

Chapter 2b: The molecular and antigenic structure of TBEV

• TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. • Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. • The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. • Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. • Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

The crystal structure of the drug targetMycobacterium tuberculosismethionyl-tRNA synthetase in complex with a catalytic intermediate

Mycobacterium tuberculosisis a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of theM. tuberculosisenzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.

Characterization and Protective Efficacy of Type III Secretion Proteins as a Broadly Protective Subunit Vaccine against Salmonella enterica Serotypes

ABSTRACT Nontyphoidal Salmonella enterica serotypes (NTS) are the leading cause of hospitalization and death due to foodborne illnesses. NTS are the costliest of the foodborne pathogens and cause ∼$4 billion annually in health care costs. In Africa, new invasive NTS are the leading cause of bacteremia, especially in HIV-positive children and adults. Current vaccines against S. enterica are not broadly protective and most are directed at the typhoid-causing serotypes, not the NTS. All S. enterica strains require two type III secretion systems (T3SS) for virulence. The T3SS needle tip protein and the first translocator are localized to the T3SS needle tip and are required for pathogenesis of S. enterica . Collectively they are 95 to 98% conserved at the amino acid sequence level among all S. enterica strains. The Salmonella pathogenicity island 1 or 2 tip and first translocator proteins were genetically fused to produce the S1 and S2 fusion proteins, respectively, as potential vaccine candidates. S1 and S2 were then characterized using spectroscopic techniques to understand their structural and biophysical properties. Formulated at the proper pH, S1, S2, or S1 plus S2 (S1S2), admixed with adjuvant, was used to immunize mice followed by a lethal challenge with S. enterica serotype Typhimurium or S. enterica serotype Enteritidis. The S1S2 formulation provided the highest protective efficacy, thus demonstrating that an S1S2 subunit vaccine can provide broad, serotype-independent protection, possibly against all S. enterica serotypes. Such a finding would be transformative in improving human health.

The Function of the COPII Gene Paralogs SEC23A and SEC23B Are Interchangeable In Vivo

SEC23 is a core component of the coat protein-complex II (COPII)-coated vesicle, which mediates transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi1-3. Mammals express 2 paralogs for SEC23 (SEC23A and SEC23B). Though the SEC23 gene duplication dates back >500 million years, both SEC23’s are ~85% identical at the amino acid sequence level. In humans, deficiency for SEC23A or SEC23B results in cranio-lenticulo-sutural dysplasia4 or congenital dyserythropoietic anemia type II (CDAII), respectively5. The disparate human syndromes and reports of secretory cargos with apparent paralog-specific dependence6,7, suggest unique functions for the two SEC23 paralogs. Here we show indistinguishable intracellular interactomes for human SEC23A and SEC23B, complementation of yeast SEC23 by both human and murine SEC23A/B paralogs, and the rescue of lethality resulting from Sec23b disruption in zebrafish by a Sec23a-expressing transgene. Finally, we demonstrate that the Sec23a coding sequence inserted into the endogenous murine Sec23b locus fully rescues the mortality and severe pancreatic phenotype previously reported with SEC23B-deficiency in the mouse8-10. Taken together, these data indicate that the disparate phenotypes of SEC23A and SEC23B deficiency likely result from evolutionary shifts in gene expression program rather than differences in protein function, a paradigm likely applicable to other sets of paralogous genes. These findings also suggest the potential for increased expression of SEC23A as a novel therapeutic approach to the treatment of CDAII, with potential relevance to other disorders due to mutations in paralogous genes.