cRegions—a tool for detecting conserved cis-elements in multiple sequence alignment of diverged coding sequences

Identifying cis-acting elements and understanding regulatory mechanisms of a gene is crucial to fully understand the molecular biology of an organism. In general, it is difficult to identify previously uncharacterised cis-acting elements with an unknown consensus sequence. The task is especially problematic with viruses containing regions of limited or no similarity to other previously characterised sequences. Fortunately, the fast increase in the number of sequenced genomes allows us to detect some of these elusive cis-elements. In this work, we introduce a web-based tool called cRegions. It was developed to identify regions within a protein-coding sequence where the conservation in the amino acid sequence is caused by the conservation in the nucleotide sequence. The cRegion can be the first step in discovering novel cis-acting sequences from diverged protein-coding genes. The results can be used as a basis for future experimental analysis. We applied cRegions on the non-structural and structural polyproteins of alphaviruses as an example and successfully detected all known cis-acting elements. In this publication and in previous work, we have shown that cRegions is able to detect a wide variety of functional elements in DNA and RNA viruses. These functional elements include splice sites, stem-loops, overlapping reading frames, internal promoters, ribosome frameshifting signals and other embedded elements with yet unknown function. The cRegions web tool is available at http://bioinfo.ut.ee/cRegions/

The enigmatic origin of papillomavirus protein domains

Almost a century has passed since the discovery of papillomaviruses. A few decades of research have given a wealth of information on the molecular biology of papillomaviruses. Several excellent studies have been performed looking at the long- and short-term evolution of these viruses. However, when and how papillomaviruses originate is still a mystery. In this study, we systematically searched the (sequenced) biosphere to find distant homologs of papillomaviral protein domains. Our data show that, even including structural information, which allows us to find deeper evolutionary relationships compared to sequence-only based methods, only half of the protein domains in papillomaviruses have relatives in the rest of the biosphere. We show that the major capsid protein L1 and the replication protein E1 have relatives in several viral families, sharing three protein domains with Polyomaviridae and Parvoviridae. However, only the E1 replication protein has connections with cellular organisms. Most likely, the papillomavirus ancestor is of marine origin, a biotope that is not very well sequenced at the present time. Nevertheless, there is no evidence as to how papillomaviruses originated and how they became vertebrate and epithelium specifi

Protein Structure-Guided Hidden Markov Models (HMMs) as A Powerful Method in the Detection of Ancestral Endogenous Viral Elements

It has been believed for a long time that the transfer and fixation of genetic material from RNA viruses to eukaryote genomes is very unlikely. However, during the last decade, there have been several cases in which “virus-to-host” gene transfer from various viral families into various eukaryotic phyla have been described. These transfers have been identified by sequence similarity, which may disappear very quickly, especially in the case of RNA viruses. However, compared to sequences, protein structure is known to be more conserved. Applying protein structure-guided protein domain-specific Hidden Markov Models, we detected homologues of the Virgaviridae capsid protein in Schizophora flies. Further data analysis supported “virus-to-host” transfer into Schizophora ancestors as a single transfer event. This transfer was not identifiable by BLAST or by other methods we applied. Our data show that structure-guided Hidden Markov Models should be used to detect ancestral virus-to-host transfers

Characterization of the Functional Activities of the Bovine Papillomavirus Type 1 E2 Protein Single-Chain Heterodimers

Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle

Did Viruses Evolve As a Distinct Supergroup from Common Ancestors of Cells?

The evolutionary origins of viruses according to marker gene phylogenies, as well as their relationships to the ancestors of host cells remains unclear. In a recent article Nasir and Caetano-Anolles reported that their genome-scale phylogenetic analyses based on genomic composition of protein structural-domains identify an ancient origin of the "viral supergroup" (Nasir et al. 2015. A phylogenomic data-driven exploration of viral origins and evolution. Sci Adv. 1(8):e1500527.). It suggests that viruses and host cells evolved independently from a universal common ancestor. Examination of their data and phylogenetic methods indicates that systematic errors likely affected the results. Reanalysis of the data with additional tests shows that small-genome attraction artifacts distort their phylogenomic analyses, particularly the location of the root of the phylogenetic tree of life that is central to their conclusions. These new results indicate that their suggestion of a distinct ancestry of the viral supergroup is not well supported by the evidence

Divalent Metal Ions Boost Effect of Nucleic Acids Delivered by Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs) are promising tools for the transfection of various substances, including nucleic acids, into cells. The aim of the current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed from CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed the effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed by the addition of the ions, without increasing cytotoxicity. We propose a novel strategy for preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, with the aim of increasing CPP-mediated transfection efficiency and the likelihood of using CPPs in clinics