Aare Abroi

Analysis of Chromatin Attachment and Partitioning Functions of Bovine Papillomavirus Type 1 E2 Protein

ABSTRACT Recent studies have suggested that the tethering of viral genomes to host cell chromosomes could provide one of the ways to achieve their nuclear retention and partitioning during extrachromosomal maintenance in dividing cells. The data we present here provide firm evidence that the partitioning of the bovine papillomavirus type 1 (BPV1) genome is dependent on the chromatin attachment process mediated by viral E2 protein and its multiple binding sites. On the other hand, the attachment of E2 and the E2-mediated tethering of reporter plasmids to host chromosomes are not necessarily sufficient for efficient partitioning, suggesting that additional E2-dependent activities might be involved in the latter process. The activity of E2 protein in chromatin attachment and partitioning is more sensitive to the point mutations in the N-terminal domain than its transactivation and replication initiation functions. Therefore, at least part of the interactions of the E2 N-terminal domain with its targets during the chromatin attachment and partitioning processes are likely to involve specific receptors not involved in transactivation and replication activities of the protein. The mutational analysis also indicates that the binding of E2 to chromatin is not achieved through interaction of linear N-terminal subsequences of the E2 protein with putative receptors. Instead, the composite surface elements of the N-terminal domain build up the receptor-binding surface of E2. In this regard, the interaction of BPV1 E2 with its chromosomal targets clearly differs from the interactions of LANA1 protein from Kaposis sarcoma-associated human herpesvirus and EBNA1 from Epstein-Barr virus with their specific receptors.

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.

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

ABSTRACT 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

ABSTRACT 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.

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.

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.

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.

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.