Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

Universal features shaping organelle gene retention

Almost all eukaryotes contain mitochondria, and many contain plastids, playing vital roles in the bioenergetic and metabolic processes that power complex life. Since their endosymbiotic origins, the genomes of both organelles have been reduced, with genes being lost or transferred to the host cell nucleus from organelle DNA (oDNA) to different extents in different species. Why some genes are retained in oDNA and some lost remains a debated question. Long-standing hypotheses include the preferential retention of genes encoding hydrophobic products and those most central to redox regulation , but quantitative testing of these and other ideas remains absent. Here we harness over 15k oDNA sequences and over 300 whole genome sequences with tools from structural biology, bioinformatics, machine learning, and Bayesian model selection to reveal the properties of protein-coding genes that shape oDNA evolution. We find striking symmetry in the features predicting mitochondrial (mtDNA) and plastid (ptDNA) gene retention. Striking symmetry exists between the two organelle types: gene retention patterns in both are predicted by the hydrophobicity of a protein product and its energetic centrality within its protein complex, with additional influences of nucleic acid and amino acid biochemistry. Supporting this generality, models trained with one organelle type successfully predict gene retention in the other, and these features also distinguish gene profiles in independent endosymbiotic relationships. The identification of these features both provide quantitative support for several existing evolutionary hypotheses, and suggest new biochemical and biophysical mechanisms influencing organelle genome evolution.

Epistatic interactions shape the interplay between beneficial alleles and gain or loss of pathways in the evolution of novel metabolism

Fitness landscapes are often invoked to interpret the effects of allele substitutions and their interactions; however, evolution also includes larger changes like gene loss and acquisition. Previous work with the methylotrophic bacterium Methylorubrum extorquens AM1 identified strongly beneficial mutations in a strain evolved to utilize a novel, Foreign pathway in place of its native central metabolic pathway for growth on methanol. These mutations were consistently beneficial, regardless of the order in which they arose. Here we extend this analysis to consider loss or acquisition of metabolic pathways by examining strains relying upon either the Native pathway, or both (‘Dual’) pathways present. Unlike in the Foreign pathway context in which they evolved, these alleles were often deleterious in these alternative genetic backgrounds, following patterns that were strongly contingent on the specific pathways and other evolved alleles present. Landscapes for these alternative pathway backgrounds altered which genotypes correspond to local fitness peaks and would restrict the set of accessible evolutionary trajectories. These epistatic interactions negatively impact the probability of maintaining multiple degenerate pathways, making it more difficult for these pathways to coevolve. Together, our results highlight the uncertainty of retaining novel functions acquired via horizontal gene transfer (HGT), and that the potential for cells to either adopt novel functions or to maintain degenerate pathways together in a genome is heavily dependent upon the underlying epistatic interactions between them.Author SummaryThe evolution of physiology in microbes has important impacts ranging from global cycling of elements to the emergence and spread of pathogens and their resistance to antibiotics. While genetic interactions between mutations in evolving lineages of microbes have been investigated, these have not included the acquisition of novel genes on elements like plasmids, and thus how these elements interact with existing alleles. The dynamics of novel gene retention are of interest from both positive (e.g., biotechnology) and negative (e.g., antimicrobial resistance) practical impacts. We find that the patterns of interactions between evolved alleles appear substantially different, and generally much less positive, when moved into novel genetic backgrounds. Additionally, these preexisting alleles were found to have strong impacts on the ability of genotypes to maintain – and in rare cases coevolve with – novel genes and pathways. These results show that even though they evolved separately, the particular alleles in a genetic background, and importantly the physiological impacts they confer, weigh heavily on whether genes for novel metabolic processes are maintained.

Biased Gene Retention in the Face of Introgression Obscures Species Relationships

Phylogenomic analyses are recovering previously hidden histories of hybridization, revealing the genomic consequences of these events on the architecture of extant genomes. We applied phylogenomic techniques and several complementary statistical tests to show that introgressive hybridization appears to have occurred between close relatives of Arabidopsis, resulting in cytonuclear discordance and impacting our understanding of species relationships in the group. The composition of introgressed and retained genes indicates that selection against incompatible cytonuclear and nuclear–nuclear interactions likely acted during introgression, whereas linkage also contributed to genome composition through the retention of ancient haplotype blocks. We also applied divergence-based tests to determine the species branching order and distinguish donor from recipient lineages. Surprisingly, these analyses suggest that cytonuclear discordance arose via extensive nuclear, rather than cytoplasmic, introgression. If true, this would mean that most of the nuclear genome was displaced during introgression whereas only a small proportion of native alleles were retained.

The matrix revolutions: towards the decoding of the plant chromatin three-dimensional reality

In recent years, we have witnessed a significant increase in studies addressing the three-dimensional (3D) chromatin organization of the plant nucleus. Important advances in chromatin conformation capture (3C)-derived and related techniques have allowed the exploration of the nuclear topology of plants with large and complex genomes, including various crops. In addition, the increase in their resolution has permitted the depiction of chromatin compartmentalization and interactions at the gene scale. These studies have revealed the highly complex mechanisms governing plant nuclear architecture and the remarkable knowledge gaps in this field. Here we discuss the state-of-the-art in plant chromosome architecture, including our knowledge of the hierarchical organization of the genome in 3D space and regarding other nuclear components. Furthermore, we highlight the existence in plants of topologically associated domain (TAD)-like structures that display striking differences from their mammalian counterparts, proposing the concept of ICONS—intergenic condensed spacers. Similarly, we explore recent advances in the study of chromatin loops and R-loops, and their implication in the regulation of gene activity. Finally, we address the impact that polyploidization has had on the chromatin topology of modern crops, and how this is related to phenomena such as subgenome dominance and biased gene retention in these organisms.

Functional specialization of human salivary glands and origins of proteins intrinsic to human saliva

SUMMARYSalivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract and serve as a diagnostic window into human disease. However, their precise organ origins remain unclear. Through transcriptomic analysis of major adult and fetal salivary glands, and integration with the saliva proteome and transcriptomes of 28+ organs, we linked human saliva proteins to their source, identified salivary gland-specific genes, and uncovered fetal- and adult-specific gene repertoires. Our results also provide new insights into the degree of gene retention during maturation and suggest that functional diversity between adult gland-types is driven by specific dosage combinations of hundreds of transcriptional regulators rather than a few gland-specific factors. Finally, we demonstrate the hitherto unrecognized heterogeneity of the human acinar cell lineage. Our results pave the way for future investigations into glandular biology and pathology, as well as saliva’s use as a diagnostic fluid.

Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochrome c oxidase

Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome c oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a cox2-null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the COX2 gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume Glycine max Cox2 could enable yeast COX2 allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.