Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids.Peer reviewe

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

The use of phage FCL-2 as an alternative to chemotherapy against columnaris disease in aquaculture

Flavobacterium columnare, the causative agent of columnaris disease in fish, causes millions of dollars of losses in the US channel catfish industry alone, not to mention aquaculture industry worldwide. Novel methods are needed for the control and treatment of bacterial diseases in aquaculture to replace traditionally used chemotherapies. A potential solution could be the use of phages, i.e., bacterial viruses, host-specific and self-enriching particles that can be can easily distributed via water flow. We examined the efficacy of phages to combat columnaris disease. A previously isolated phage, FCL-2, infecting F. columnare, was characterized by sequencing. The 47 142 bp genome of the phage had G + C content of 30.2%, and the closest similarities regarding the structural proteins were found in Cellulophaga phage phiSM. Under controlled experimental conditions, two host fish species, rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), were used to study the success of phage therapy to prevent F. columnare infections. The survival of both fish species was significantly higher in the presence of the phage. Hundred percent of the zebrafish and 50 % of the rainbow trout survived in the phage treatment (survival without phage 0 % and 8.3 %, respectively). Most importantly, the rainbow trout population was rescued from infection by a single addition of the phage into the water in a flow-through fish tank system. Thus, F. columnare could be used as a model system to test the benefits and risks of phage therapy on a larger scale.Peer reviewe

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 viruse

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.Peer reviewe

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

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

Evidence for a Non-Catalytic Ion-Binding Site in Multiple RNA-Dependent RNA Polymerases

Peer reviewe

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.Peer reviewe

Ice nucleation by viruses and their potential for cloud glaciation

In order to effectively predict the formation of ice in clouds we need to know which subsets of aerosol particles are effective at nucleating ice, how they are distributed and where they are from. A large proportion of ice-nucleating particles (INPs) in many locations are likely of biological origin, and some INPs are extremely small, being just tens of nanometres in size. The identity and sources of such INPs are not well characterized. Here, we show that several different types of virus particles can nucleate ice, with up to about 1 in 20 million virus particles able to nucleate ice at -20 degrees C. In terms of the impact on cloud glaciation, the ice-nucleating ability (the fraction which are ice nucleation active as a function of temperature) taken together with typical virus particle concentrations in the atmosphere leads to the conclusion that virus particles make a minor contribution to the atmospheric ice-nucleating particle population in the terrestrial-influenced atmosphere. However, they cannot be ruled out as being important in the remote marine atmosphere. It is striking that virus particles have an ice-nucleating activity, and further work should be done to explore other types of viruses for both their ice-nucleating potential and to understand the mechanism by which viruses nucleate ice.Peer reviewe