Detection of a major Lynch Syndrome-causing MLH1 founder variant in a large-scale genotyped cohort.

Some 50% of Finnish Lynch Syndrome (LS) cases are caused by a founder variant in MLH1, in which the entire exon 16 has been lost due to an Alu-mediated recombination event. We piloted detecting the variant in FinnGen, a large genotyped cohort comprising approximately 10% of the current Finnish population, and validated the MLH1 founder variant status of identified individuals residing in the Central Finland Biobank catchment area. A consensus sequence flanking the deletion was identified in whole genome sequences of six LS individuals with the founder variant. Genotype data of 212,196 individuals was queried for regional matches to the consensus sequence. Enrichment of cancer and age at cancer onset was compared between matching and non-matching individuals. Variant status was validated for a subset of the identified individuals using a polymerase chain reaction assay. Allelic matches in a chosen target region was detected in 348 individuals, with 89 having a cancer diagnosis (Bonferroni-adjusted p-value = 1), 20 a familial cancer history (p-adj. < .001), with mean age of onset of cancer being 53.6 years (p-adj. = .002). Eighteen of potential variant carriers had been sampled by the Central Finland Biobank, of which four (22%) were validated as true variant carriers. The workflow we have employed identifies MLH1 exon 16 deletion variant carriers from population-wide SNP genotyping data. An alternative design will be sought to limit false positive findings. Large genotyped cohorts provide a potential resource for identification and prevention of hereditary cancer.

Relevance of the bacteriophage adherence to mucus model for Pseudomonas aeruginosa phages.

Pseudomonas aeruginosa infections are getting increasingly serious as antimicrobial resistance spreads. Phage therapy may be a solution to the problem, especially if improved by current advances on phage-host studies. As a mucosal pathogen, we hypothesize that P. aeruginosa and its phages are linked to the bacteriophage adherence to mucus (BAM) model. This means that phage-host interactions could be influenced by mucin presence, impacting the success of phage infections on the P. aeruginosa host and consequently leading to the protection of the metazoan host. By using a group of four different phages, we tested three important phenotypes associated with the BAM model: phage binding to mucin, phage growth in mucin-exposed hosts, and the influence of mucin on CRISPR immunity of the bacterium. Three of the tested phages significantly bound to mucin, while two had improved growth rates in mucin-exposed hosts. Improved phage growth was likely the result of phage exploitation of mucin-induced physiological changes in the host. We could not detect CRISPR activity in our system but identified two putative anti-CRISPR proteins coded by the phage. Overall, the differential responses seen for the phages tested show that the same bacterial species can be targeted by mucosal-associated phages or by phages not affected by mucus presence. In conclusion, the BAM model is relevant for phage-bacterium interactions in P. aeruginosa, opening new possibilities to improve phage therapy against this important pathogen by considering mucosal interaction dynamics.IMPORTANCESome bacteriophages are involved in a symbiotic relationship with animals, in which phages held in mucosal surfaces protect them from invading bacteria. Pseudomonas aeruginosa is one of the many bacterial pathogens threatening humankind during the current antimicrobial resistance crisis. Here, we have tested whether P. aeruginosa and its phages are affected by mucosal conditions. We discovered by using a collection of four phages that, indeed, mucosal interaction dynamics can be seen in this model. Three of the tested phages significantly bound to mucin, while two had improved growth rates in mucin-exposed hosts. These results link P. aeruginosa and its phages to the bacteriophage adherence to the mucus model and open opportunities to explore this to improve phage therapy, be it by exploiting the phenotypes detected or by actively selecting mucosal-adapted phages for treatment.

Genome-wide somatic mutation analysis of sinonasal adenocarcinoma with and without wood dust exposure.

BACKGROUND: Sinonasal adenocarcinoma is a rare cancer, encompassing two different entities, the intestinal-type sinonasal adenocarcinoma (ITAC) and the non-intestinal-type sinonasal adenocarcinoma (non-ITAC). Occurrence of ITAC is strongly associated with exposure to hardwood dusts. In countries with predominant exposure to softwood dust the occurrence of sinonasal adenocarcinomas is lower and the relative amount of non-ITACs to ITACs is higher. The molecular mechanisms behind the tumorigenic effects of wood dust remain largely unknown. METHODS: We carried out whole-genome sequencing of formalin-fixed paraffin-embedded (FFPE) samples of sinonasal adenocarcinomas from ten wood dust-exposed and six non-exposed individuals, with partial tobacco exposure data. Sequences were analyzed for the presence of mutational signatures matching COSMIC database signatures. Driver mutations and CN variant regions were characterized. RESULTS: Mutation burden was higher in samples of wood dust-exposed patients (p = 0.016). Reactive oxygen species (ROS) damage-related mutational signatures were almost exclusively identified in ITAC subtype samples (p = 0.00055). Tobacco smoke mutational signatures were observed in samples of patients with tobacco exposure or missing information, but not in samples from non-exposed patients. A tetraploidy copy number (CN) signature was enriched in ITAC subtype (p = 0.042). CN variation included recurrent gains in COSMIC Cancer Gene Census genes TERT, SDHA, RAC1, ETV1, PCM1, and MYC. Pathogenic variants were observed most frequently in TP53, NF1, CHD2, BRAF, APC, and LRP1B. Driver mutations and copy number gains did not segregate by subtype. CONCLUSIONS: Our analysis identified distinct mutational characteristics in ITAC and non-ITAC. Mutational signature analysis may eventually become useful for documentation of occupation-related cancer, while the exact mechanisms behind wood dust-driven carcinogenesis remain elusive. The presence of homologous recombination deficiency signatures implies a novel opportunity for treatment, but further studies are needed.

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

Cellular state landscape and herpes simplex virus type 1 infection progression are connected.

Prediction, prevention and treatment of virus infections require understanding of cell-to-cell variability that leads to heterogenous disease outcomes, but the source of this heterogeneity has yet to be clarified. To study the multimodal response of single human cells to herpes simplex virus type 1 (HSV-1) infection, we mapped high-dimensional viral and cellular state spaces throughout the infection using multiplexed imaging and quantitative single-cell measurements of viral and cellular mRNAs and proteins. Here we show that the high-dimensional cellular state scape can predict heterogenous infections, and cells move through the cellular state landscape according to infection progression. Spatial information reveals that infection changes the cellular state of both infected cells and of their neighbors. The multiplexed imaging of HSV-1-induced cellular modifications links infection progression to changes in signaling responses, transcriptional activity, and processing bodies. Our data show that multiplexed quantification of responses at the single-cell level, across thousands of cells helps predict infections and identify new targets for antivirals.

A structural dendrogram of the actinobacteriophage major capsid proteins provides important structural insights into the evolution of capsid stability.

Many double-stranded DNA viruses, including tailed bacteriophages (phages) and herpesviruses, use the HK97-fold in their major capsid protein to make the capsomers of the icosahedral viral capsid. After the genome packaging at near-crystalline densities, the capsid is subjected to a major expansion and stabilization step that allows it to withstand environmental stresses and internal high pressure. Several different mechanisms for stabilizing the capsid have been structurally characterized, but how these mechanisms have evolved is still not understood. Using cryo-EM structure determination of 10 capsids, structural comparisons, phylogenetic analyses, and Alphafold predictions, we have constructed a detailed structural dendrogram describing the evolution of capsid structural stability within the actinobacteriophages. We show that the actinobacteriophage major capsid proteins can be classified into 15 groups based upon their HK97-fold.

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution.

The origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipid-containing ssDNA temperate bacteriophage ΦCjT23, which infects Flavobacterium sp. (Bacteroidetes). We report ΦCjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses with a major capsid protein composed of two upright ß-sandwiches. The minimalistic structure of ΦCjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both ΦCjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and a shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.

Sex-specific familial aggregation of cancers in Finland.

Despite the fact that the effect of sex on the occurrence of cancers has been studied extensively, it remains unclear whether sex modifies familial aggregation of cancers. We explored sex-specific familial aggregation of cancers in a large population-based historical cohort study. We combined cancer and population registry data, inferring familial relationships from birth municipality-surname-sex (MNS) combinations. Our data consisted of 391,529 incident primary cancers in 377,210 individuals with 319,872 different MNS combinations. Cumulative sex-specific numbers of cancers were compared to expected cumulative incidence. Familial cancer risks were similar between the sexes in our population-wide analysis. Families with concordant cancer in both sexes exhibited similar sex-specific cancer risks. However, some families had exceptionally high sex-specific cumulative cancer incidence. We identified six families with exceptionally strong aggregation in males: three families with thyroid cancer (ratio between observed and expected incidence 184.6; 95% credible interval (95% CI) 33.1-1012.7, 173.4 (95% CI 65.4-374.3), and 161.4 (95% CI 29.6-785.7), one with stomach (ratio 14.4 (95% CI 6.9-37.2)), colon (ratio 15.5 (95% CI 5.7-56.3)) cancers and one with chronic lymphocytic leukaemia (ratio 33.5 (95% CI 17.2-207.6)). Our results imply that familial aggregation of cancers shows no sex-specific preference. However, the atypical sex-specific aggregation of stomach cancer, colon cancer, thyroid cancer and chronic lymphocytic leukaemia in certain families is difficult to fully explain with present knowledge of possible causes, and could yield useful knowledge if explored further.

Mucin induces CRISPR-Cas defense in an opportunistic pathogen.

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies obtain at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the phage-bacterium interactions on mucosal surfaces may select for the diversification of bacterial immune systems.

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.

Genetic and Epigenetic Characteristics of Inflammatory Bowel Disease-Associated Colorectal Cancer.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder associated with an elevated risk of colorectal cancer (CRC). IBD-associated CRC (IBD-CRC) may represent a distinct pathway of tumorigenesis compared to sporadic CRC (sCRC). Our aim was to comprehensively characterize IBD-associated tumorigenesis integrating multiple high-throughput approaches, and to compare the results with in-house data sets from sCRCs. METHODS: Whole-genome sequencing, single nucleotide polymorphism arrays, RNA sequencing, genome-wide methylation analysis, and immunohistochemistry were performed using fresh-frozen and formalin-fixed tissue samples of tumor and corresponding normal tissues from 31 patients with IBD-CRC. RESULTS: Transcriptome-based tumor subtyping revealed the complete absence of canonical epithelial tumor subtype associated with WNT signaling in IBD-CRCs, dominated instead by mesenchymal stroma-rich subtype. Negative WNT regulators AXIN2 and RNF43 were strongly down-regulated in IBD-CRCs and chromosomal gains at HNF4A, a negative regulator of WNT-induced epithelial-mesenchymal transition (EMT), were less frequent compared to sCRCs. Enrichment of hypomethylation at HNF4α binding sites was detected solely in sCRC genomes. PIGR and OSMR involved in mucosal immunity were dysregulated via epigenetic modifications in IBD-CRCs. Genome-wide analysis showed significant enrichment of noncoding mutations to 5'untranslated region of TP53 in IBD-CRCs. As reported previously, somatic mutations in APC and KRAS were less frequent in IBD-CRCs compared to sCRCs. CONCLUSIONS: Distinct mechanisms of WNT pathway dysregulation skew IBD-CRCs toward mesenchymal tumor subtype, which may affect prognosis and treatment options. Increased OSMR signaling may favor the establishment of mesenchymal tumors in patients with IBD.

Structure Unveils Relationships between RNA Virus Polymerases.

RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization.

Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System.

CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules.IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader's genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system "borrows" acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers.

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.

Prophages and Past Prophage-Host Interactions Revealed by CRISPR Spacer Content in a Fish Pathogen.

The role of prophages in the evolution, diversification, or virulence of the fish pathogen Flavobacterium columnare has not been studied thus far. Here, we describe a functional spontaneously inducing prophage fF4 from the F. columnare type strain ATCC 23463, which is not detectable with commonly used prophage search methods. We show that this prophage type has a global distribution and is present in strains isolated from Finland, Thailand, Japan, and North America. The virions of fF4 are myoviruses with contractile tails and infect only bacterial strains originating from Northern Finland. The fF4 resembles transposable phages by similar genome organization and several gene orthologs. Additional bioinformatic analyses reveal several species in the phylum Bacteroidetes that host a similar type of putative prophage, including bacteria that are important animal and human pathogens. Furthermore, a survey of F. columnare Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers indicate a shared evolutionary history between F. columnare strains and the fF4 phage, and another putative prophage in the F. columnare strain ATCC 49512, named p49512. First, CRISPR spacer content from the two CRISPR loci (types II-C and VI-B) of the fF4 lysogen F. columnare ATCC 23463 revealed a phage terminase protein-matching spacer in the VI-B locus. This spacer is also present in two Chinese F. columnare strains. Second, CRISPR analysis revealed four F. columnare strains that contain unique spacers targeting different regions of the putative prophage p49512 in the F. columnare strain ATCC 49512, despite the geographical distance or genomovar of the different strains. This suggests a common ancestry for the F. columnare prophages and different host strains.

Superimposition of Viral Protein Structures: A Means to Decipher the Phylogenies of Viruses.

Superimposition of protein structures is key in unravelling structural homology across proteins whose sequence similarity is lost. Structural comparison provides insights into protein function and evolution. Here, we review some of the original findings and thoughts that have led to the current established structure-based phylogeny of viruses: starting from the original observation that the major capsid proteins of plant and animal viruses possess similar folds, to the idea that each virus has an innate "self". This latter idea fueled the conceptualization of the PRD1-adenovirus lineage whose members possess a major capsid protein (innate "self") with a double jelly roll fold. Based on this approach, long-range viral evolutionary relationships can be detected allowing the virosphere to be classified in four structure-based lineages. However, this process is not without its challenges or limitations. As an example of these hurdles, we finally touch on the difficulty of establishing structural "self" traits for enveloped viruses showcasing the coronaviruses but also the power of structure-based analysis in the understanding of emerging viruses.

Adapting a Phage to Combat Phage Resistance.

Phage therapy is becoming a widely recognized alternative for fighting pathogenic bacteria due to increasing antibiotic resistance problems. However, one of the common concerns related to the use of phages is the evolution of bacterial resistance against the phages, putatively disabling the treatment. Experimental adaptation of the phage (phage training) to infect a resistant host has been used to combat this problem. Yet, there is very little information on the trade-offs of phage infectivity and host range. Here we co-cultured a myophage FCV-1 with its host, the fish pathogen Flavobacterium columnare, in lake water and monitored the interaction for a one-month period. Phage resistance was detected within one day of co-culture in the majority of the bacterial isolates (16 out of the 18 co-evolved clones). The primary phage resistance mechanism suggests defense via surface modifications, as the phage numbers rose in the first two days of the experiment and remained stable thereafter. However, one bacterial isolate had acquired a spacer in its CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas locus, indicating that also CRISPR-Cas defense was employed in the phage-host interactions. After a week of co-culture, a phage isolate was obtained that was able to infect 18 out of the 32 otherwise resistant clones isolated during the experiment. Phage genome sequencing revealed several mutations in two open reading frames (ORFs) likely to be involved in the regained infectivity of the evolved phage. Their location in the genome suggests that they encode tail genes. Characterization of this evolved phage, however, showed a direct cost for the ability to infect several otherwise resistant clones-adsorption was significantly lower than in the ancestral phage. This work describes a method for adapting the phage to overcome phage resistance in a fish pathogenic system.

Complete Genome Sequence of Fish Pathogen Flavobacterium columnare Strain B185, Originating from Finland.

We report a complete genome sequence of a Finnish isolate of the fish pathogen Flavobacterium columnare Using PacBio RS II sequencing technology, the complete circular genome of F. columnare strain B185 with 3,261,404 bp was obtained.

Structural comparison strengthens the higher-order classification of proteases related to chymotrypsin.

Specific cleavage of proteins by proteases is essential for several cellular, physiological, and viral processes. Chymotrypsin-related proteases that form the PA clan in the MEROPS classification of proteases is one of the largest and most diverse group of proteases. The PA clan comprises serine proteases from bacteria, eukaryotes, archaea, and viruses and chymotrypsin-related cysteine proteases from positive-strand RNA viruses. Despite low amino acid sequence identity, all PA clan proteases share a conserved double ß-barrel structure. Using an automated structure-based hierarchical clustering method, we identified a common structural core of 72 amino acid residues for 143 PA clan proteases that represent 12 protein families and 11 subfamilies. The identified core is located around the catalytic site between the two ß-barrels and resembles the structures of the smallest PA clan proteases. We constructed a structure-based distance tree derived from the properties of the identified common core. Our structure-based analyses support the current classification of these proteases at the subfamily level and largely at the family level. Structural alignment and structure-based distance trees could thus be used for directing objective classification of PA clan proteases and to strengthen their higher order classification. Our results also indicate that the PA clan proteases of positive-strand RNA viruses are related to cellular heat-shock proteases, which suggests that the exchange of protease genes between viruses and cells might have occurred more than once.

Ribosome profiles and riboproteomes of healthy and Potato virus A- and Agrobacterium-infected Nicotiana benthamiana plants.

Nicotiana benthamiana is an important model plant for plant-microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants.