Viral trans-factor independent replication of human papillomavirus genomes

<p>Abstract</p> <p>Background</p> <p>Papillomaviruses (PVs) establish a persistent infection in the proliferating basal cells of the epithelium. The viral genome is replicated and maintained as a low-copy nuclear plasmid in basal keratinocytes. Bovine and human papillomaviruses (BPV and HPV) are known to utilize two viral proteins; E1, a DNA helicase, and E2, a transcription factor, which have been considered essential for viral DNA replication. However, growing evidence suggests that E1 and E2 are not entirely essential for stable replication of HPV.</p> <p>Results</p> <p>Here we report that multiple HPV16 mutants, lacking either or both E1 and E2 open reading frame (ORFs) and the long control region (LCR), still support extrachromosomal replication. Our data clearly indicate that HPV16 has a mode of replication, independent of viral trans-factors, E1 and E2, which is achieved by origin activity located outside of the LCR.</p

Primary Raynaud's phenomenon in an infant: a case report and review of literature

Raynaud's phenomenon (RP) is an extremely unusual finding in early infancy. In the present report we describe a one-month-old previously healthy male infant who presented with unilateral acrocyanosis. Although infantile acrocyanosis is known to be a benign and self-resolving condition, it is generally bilateral and symmetric. The unilateral nature of the acrocyanosis was an atypical finding in this infant. Consequently, he was closely monitored to evaluate the progression of his acrocyanosis. Based on his benign clinical course and failure to demonstrate other etiologies contributing to his acrocyanosis, he was diagnosed to have primary RP. Due to the rarity of RP in children, we review the progress in understanding the pathophysiology, epidemiology and management of RP and additionally discuss the differential diagnosis of unilateral and bilateral acrocyanosis in infants

Atomic-accuracy prediction of protein loop structures through an RNA-inspired ansatz

Consistently predicting biopolymer structure at atomic resolution from sequence alone remains a difficult problem, even for small sub-segments of large proteins. Such loop prediction challenges, which arise frequently in comparative modeling and protein design, can become intractable as loop lengths exceed 10 residues and if surrounding side-chain conformations are erased. This article introduces a modeling strategy based on a 'stepwise ansatz', recently developed for RNA modeling, which posits that any realistic all-atom molecular conformation can be built up by residue-by-residue stepwise enumeration. When harnessed to a dynamic-programming-like recursion in the Rosetta framework, the resulting stepwise assembly (SWA) protocol enables enumerative sampling of a 12 residue loop at a significant but achievable cost of thousands of CPU-hours. In a previously established benchmark, SWA recovers crystallographic conformations with sub-Angstrom accuracy for 19 of 20 loops, compared to 14 of 20 by KIC modeling with a comparable expenditure of computational power. Furthermore, SWA gives high accuracy results on an additional set of 15 loops highlighted in the biological literature for their irregularity or unusual length. Successes include cis-Pro touch turns, loops that pass through tunnels of other side-chains, and loops of lengths up to 24 residues. Remaining problem cases are traced to inaccuracies in the Rosetta all-atom energy function. In five additional blind tests, SWA achieves sub-Angstrom accuracy models, including the first such success in a protein/RNA binding interface, the YbxF/kink-turn interaction in the fourth RNA-puzzle competition. These results establish all-atom enumeration as a systematic approach to protein structure that can leverage high performance computing and physically realistic energy functions to more consistently achieve atomic resolution.Comment: Identity of four-loop blind test protein and parts of figures 5 have been omitted in this preprint to ensure confidentiality of the protein structure prior to its public releas

Structural basis for the initiation of eukaryotic transcription-coupled DNA repair

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II–CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II–Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation

Crowdsourced science: sociotechnical epistemology in the e-research paradigm

Recent years have seen a surge in online collaboration between experts and amateurs on scientific research. In this article, we analyse the epistemological implications of these crowdsourced projects, with a focus on Zooniverse, the world’s largest citizen science web portal. We use quantitative methods to evaluate the platform’s success in producing large volumes of observation statements and high impact scientific discoveries relative to more conventional means of data processing. Through empirical evidence, Bayesian reasoning, and conceptual analysis, we show how information and communication technologies enhance the reliability, scalability, and connectivity of crowdsourced e-research, giving online citizen science projects powerful epistemic advantages over more traditional modes of scientific investigation. These results highlight the essential role played by technologically mediated social interaction in contemporary knowledge production. We conclude by calling for an explicitly sociotechnical turn in the philosophy of science that combines insights from statistics and logic to analyse the latest developments in scientific research

In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain

The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes cell transformation by binding and activating the platelet-derived growth factor beta receptor (PDGF beta R). In order to productively interact with the receptor, it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers, and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer; indeed, mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable alpha-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation demonstrate for the first time that the TM domain of E5 forms a strong dimer with a standard state free energy of dissociation of 5.0 kcal mol(-1). We have used our new results to interpret existing models of E5 dimer formation and provide a direct link between TM helix interactions and E5 function

De novo protein structure determination from near-atomic-resolution cryo-EM maps

We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-Å resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps