Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor.

Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases-N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11-as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central "hub" protein that regulates the activity of at least seven methyltransferases.

Conservation of the E8 CDS of the E8

Papillomaviridae are small dsDNA viruses with a limited coding capacity. To fulfill all of the functional requirements for propagation and spreading, papillomaviruses use double coding and alternative protein isoforms. E8 ^ E2 is an alternative E2 protein isoform that is generated by fusing the short E8 CDS that completely overlaps E1 to the 'hinge' and the DNA-binding region of the E2 protein via alternative transcription/splicing. The papillomaviruses in which E8 ^ E2 mRNA sequences have been described exhibit a sparse phylogenomic distribution. Thus, it is not clear whether E8 ^ E2 is an ancestral protein that has not been described for other papillomavirus types or whether it randomly appears because of the conservation of the E1 protein and occurs only coincidentally. We searched for potential E8 coding sequences in a non-redundant set of papillomaviruses and applied SynPlot2 and an in-house-developed algorithm (cRegions) to determine the most plausible of the above-mentioned scenarios. Beginning with nine experimentally described E8 ^ E2 mRNAs, we predicted the potential E8 CDSs for more than 300 mammalian papillomavirus genomes. According to our analysis, E8 ^ E2 is not a result of E1 coding and represents a protein in its own right, and it most likely has an ancestral origin that precedes the divergence of major mammalian papillomavirus genera.

SARS-CoV-2 clade dynamics and their associations with hospitalisations during the first two years of the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic was characterised by rapid waves of disease, carried by the emergence of new and more infectious SARS-CoV-2 virus variants. How the pandemic unfolded in various locations during its first two years has yet to be sufficiently covered. To this end, here we are looking at the circulating SARS-CoV-2 variants, their diversity, and hospitalisation rates in Estonia in the period from March 2000 to March 2022. METHODS: We sequenced a total of 27,550 SARS-CoV-2 samples in Estonia between March 2020 and March 2022. High-quality sequences were genotyped and assigned to Nextstrain clades and Pango lineages. We used regression analysis to determine the dynamics of lineage diversity and the probability of clade-specific hospitalisation stratified by age and sex. RESULTS: We successfully sequenced a total of 25,375 SARS-CoV-2 genomes (or 92%), identifying 19 Nextstrain clades and 199 Pango lineages. In 2020 the most prevalent clades were 20B and 20A. The various subsequent waves of infection were driven by 20I (Alpha), 21J (Delta) and Omicron clades 21K and 21L. Lineage diversity via the Shannon index was at its highest during the Delta wave. About 3% of sequenced SARS-CoV-2 samples came from hospitalised individuals. Hospitalisation increased markedly with age in the over-forties, and was negligible in the under-forties. Vaccination decreased the odds of hospitalisation in over-forties. The effect of vaccination on hospitalisation rates was strongly dependent upon age but was clade-independent. People who were infected with Omicron clades had a lower hospitalisation likelihood in age groups of forty and over than was the case with pre-Omicron clades regardless of vaccination status. CONCLUSIONS: COVID-19 disease waves in Estonia were driven by the Alpha, Delta, and Omicron clades. Omicron clades were associated with a substantially lower hospitalisation probability than pre-Omicron clades. The protective effect of vaccination in reducing hospitalisation likelihood was independent of the involved clade.

Are viruses a source of new protein folds for organisms? - Virosphere structure space and evolution.

A crucially important part of the biosphere - the virosphere - is too often overlooked. Inclusion of the virosphere into the global picture of protein structure space reveals that 63 protein domain superfamilies in viruses do not have any structural and evolutionary relatives in modern cellular organisms. More than half of these have functions which are not virus-specific and thus might be a source of new folds and functions for cellular life. The number of viruses on the planet exceeds that of cells by an order of magnitude and viruses evolve up to six orders of magnitude faster. As a result, cellular species are subject to a constitutive 'flow-through' of new viral genetic material. Due to this and the relaxed evolutionary constraints in viruses, the transfer of domains between host-to-virus could be a mechanism for accelerated protein evolution. The virosphere could be an engine for the genesis of protein structures, and may even have been so before the last universal common ancestor of cellular life.

Risk of SARS-CoV-2 infection and hospitalization in individuals with natural, vaccine-induced and hybrid immunity: a retrospective population-based cohort study from Estonia.

A large proportion of the world's population has some form of immunity against SARS-CoV-2, through either infection ('natural'), vaccination or both ('hybrid'). This retrospective cohort study used data on SARS-CoV-2, vaccination, and hospitalization from national health system from February 2020 to June 2022 and Cox regression modelling to compare those with natural immunity to those with no (Cohort1, n = 94,982), hybrid (Cohort2, n = 47,342), and vaccine (Cohort3, n = 254,920) immunity. In Cohort 1, those with natural immunity were at lower risk for infection during the Delta (aHR 0.17, 95%CI 0.15-0.18) and higher risk (aHR 1.24, 95%CI 1.18-1.32) during the Omicron period than those with no immunity. Natural immunity conferred substantial protection against COVID-19-hospitalization. Cohort 2-in comparison to natural immunity hybrid immunity offered strong protection during the Delta (aHR 0.61, 95%CI 0.46-0.80) but not the Omicron (aHR 1.05, 95%CI 0.93-1.1) period. COVID-19-hospitalization was extremely rare among individuals with hybrid immunity. In Cohort 3, individuals with vaccine-induced immunity were at higher risk than those with natural immunity for infection (Delta aHR 4.90, 95%CI 4.48-5.36; Omicron 1.13, 95%CI 1.06-1.21) and hospitalization (Delta aHR 7.19, 95%CI 4.02-12.84). These results show that risk of infection and severe COVID-19 are driven by personal immunity history and the variant of SARS-CoV-2 causing infection.

Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery.

Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP-NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide-cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

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.

SARS-CoV-2 dual infection with Delta and Omicron variants in an immunocompetent host: a case report.

Despite the high number of SARS-CoV-2 infections, only a few cases of dual infection have been reported. Here, we describe a case of COVID-19 caused simultaneously by Delta and Omicron variants in an immunocompetent individual during the early emergence of Omicron variant. A 73-year-old man was hospitalized with suspected acute coronary syndrome and a positive test result for SARS-CoV-2 RNA was received during routine testing at the hospital. He experienced mild symptoms of COVID-19 and was discharged on the ninth day. We sequenced the SARS-CoV-2 whole genome from the sample obtained on admission. The viral sequence was classified as PANGO lineage B.1.1.10 by the Galaxy pipeline; however, on detailed manual analysis, we identified the presence of both Delta and Omicron variants. After excluding the possibilities of a recombinant virus or contamination in the sample, we confirmed the presence of dual infection in this patient. We highlight that dual infections with SARS-CoV-2 may be more common than expected but are difficult to detect during the waves of one dominant variant.

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/.

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.

The Common Partner of Several Methyltransferases TRMT112 Regulates the Expression of N6AMT1 Isoforms in Mammalian Cells.

Methylation is a widespread modification occurring in DNA, RNA and proteins. The N6AMT1 (HEMK2) protein has DNA N6-methyladenine as well as the protein glutamine and histone lysine methyltransferase activities. The human genome encodes two different isoforms of N6AMT1, the major isoform and the alternatively spliced isoform, where the substrate binding motif is missing. Several RNA methyltransferases involved in ribosome biogenesis, tRNA methylation and translation interact with the common partner, the TRMT112 protein. In this study, we show that TRMT112 regulates the expression of N6AMT1 isoforms in mammalian cells. Both isoforms are equally expressed on mRNA level, but only isoform 1 is detected on the protein level in human cells. We show that the alternatively spliced isoform is not able to interact with TRMT112 and when translated, is rapidly degraded from the cells. This suggests that TRMT112 is involved in cellular quality control ensuring that N6AMT1 isoform with missing substrate binding domain is eliminated from the cells. The down-regulation of TRMT112 does not affect the N6AMT1 protein levels in cells, suggesting that the two proteins of TRMT112 network, WBSCR22 and N6AMT1, are differently regulated by their common cofactor.

The Third Annual Meeting of the European Virus Bioinformatics Center.

The Third Annual Meeting of the European Virus Bioinformatics Center (EVBC) took place in Glasgow, United Kingdom, 28-29 March 2019. Virus bioinformatics has become central to virology research, and advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks, being successfully used to detect, control, and treat infections of humans and animals. This active field of research has attracted approximately 110 experts in virology and bioinformatics/computational biology from Europe and other parts of the world to attend the two-day meeting in Glasgow to increase scientific exchange between laboratory- and computer-based researchers. The meeting was held at the McIntyre Building of the University of Glasgow; a perfect location, as it was originally built to be a place for "rubbing your brains with those of other people", as Rector Stanley Baldwin described it. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The meeting featured eight invited and twelve contributed talks, on the four main topics: (1) systems virology, (2) virus-host interactions and the virome, (3) virus classification and evolution and (4) epidemiology, surveillance and evolution. Further, the meeting featured 34 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting.

Nuclear myosin 1 associates with papillomavirus E2 regulatory protein and influences viral replication.

Nuclear myosin 1c (NM1) associates with RNA polymerases and is a partner in the chromatin remodeling complex B-WICH. This complex, which also contains WSTF and SNF2h proteins, is involved in transcriptional regulation. We report herein that papillomavirus protein E2 binds to NM1 and co-precipitates with the WSTF and SNF2h proteins. Our data suggest that E2 associates with the cellular B-WICH complex through binding to NM1. E2 and NM1 associate via their N-terminal domains and this interaction is ATP dependent. The cellular multifunctional protein Brd4 and beta-actin are also present in the NM1-E2 complex. NM1 downregulation by siRNA increases the replication of the BPV1 and HPV5 genomes but does not affect HPV18 genome replication. These results suggest that the B-WICH complex may play a role in the papillomavirus life cycle through NM1 and E2 protein interaction.

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 specific.

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-Anollés 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.

A protein domain-based view of the virosphere-host relationship.

Despite being an important and inseparable part of the biosphere, viruses are too often overlooked in several life sciences, including evolutionary biology, systems biology, and non-marine ecology. In this review, a protein domain-based view of viral proteomes, the proteomes of other organisms and the overlap between them is presented. The data show that in many viral species, viral proteins are not very well annotated with protein domains. Compared with viral proteomes, cellular proteomes are covered quite uniformly with respect to protein domains and show higher coverage. A tremendous number of virally coded domains exist; in fact, the number of protein domains in the characterised virosphere is approaching that found in Archaea, a well-accepted superkingdom. Proteins encoded by viruses contain virosphere-specific domains (i.e., not found in cellular proteomes) and/or many domains shared by viral and cellular proteomes. Virosphere-specific domains are structurally peculiar with respect to different structural measures, making them a clear source of structural and functional novelty. Viral families with RNA genomes tend to harbour more virosphere-specific domains than other viruses. Interestingly, host range preferences of different viral classes are, for the most part, not reflected by domains shared between viruses and different superkingdoms. The role of viruses in the genesis of the cellular domain repertoire is reviewed to bring them more confidently and firmly into the larger biological picture.

Characterization of the nuclear matrix targeting sequence (NMTS) of the BPV1 E8/E2 protein--the shortest known NMTS.

Technological advantages in sequencing and proteomics have revealed the remarkable diversity of alternative protein isoforms. Typically, the localization and functions of these isoforms are unknown and cannot be predicted. Also the localization signals leading to particular subnuclear compartments have not been identified and thus, predicting alternative functions due to alternative subnuclear localization is limited only to very few subnuclear compartments. Knowledge of the localization and function of alternative protein isoforms allows for a greater understanding of cellular complexity. In this article, we characterize a short and well-defined signal targeting the bovine papillomavirus type 1 E8/E2 protein to the nuclear matrix. The targeting signal comprises the peptide coded by E8 ORF, which is spliced together with part of the E2 ORF to generate the E8/E2 mRNA. Localization to the nuclear matrix correlates well with the transcription repression activities of E8/E2; a single point mutation directs the E8/E2 protein into the nucleoplasm, and transcription repression activity is lost. Our data prove that adding as few as ˜10 amino acids by alternative transcription/alternative splicing drastically alters the function and subnuclear localization of proteins. To our knowledge, E8 is the shortest known nuclear matrix targeting signal.

Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo.

Papillomaviruses are small DNA viruses that induce epithelial lesions in their host. The viral life cycle is regulated by the family of proteins encoded by the E2 open reading frame. In addition to the full-length E2 protein, the BPV-1 genome encodes two truncated E2 proteins, E2C and E8/E2, which maintain the DNA-binding-dimerization domains, but lack the activation domain. Heterodimers formed between the full-length E2 and truncated E2 proteins serve as activators of E2-dependent transcription and papillomavirus DNA replication. We show that the single activation domain of E2 is sufficient for interaction with viral helicase E1 and for initiation of DNA replication from different papillomavirus origins. Single-chain E2 heterodimer is able to activate papillomavirus DNA replication in the context of entire BPV genome in the absence of other E2 proteins. These data suggest that E2 heterodimers with single activation domain are functional in initiation of papillomavirus replication in vivo.

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.

Episomal maintenance of plasmids with hybrid origins in mouse cells.

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable maintenance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13% in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells.