Aggregate and spatial distributions of DNA palindromes and their applications to replication origins prediction in some viral genomes

Ph.DDOCTOR OF PHILOSOPH

Palindrome distributions and their applications

Master'sMASTER OF SCIENC

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis

Genetic approaches to the study of coronavirus replication and pathogenesis

The recently developed coronavirus reverse genetic systems have been a tremendous asset for improving our understanding of the viruses' complex replication strategy, pathogenesis, mechanisms of host-range expansion, and in the development of anti-viral therapies. We completed two studies using coronavirus infectious clones. The first evaluated a severe acute respiratory syndrome coronavirus (SARS-CoV) vaccine to protect against an antigenically divergent strain. The second study determined the requirement for proteolytic processing of a highly conserved region of the replicase polyprotein for efficient replication. Ideally, a SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N) as candidate vaccines, we tested their ability to protect young and senescent mice when challenged with homologous and heterologous SARS-CoV strains. The novel heterologous SARS-CoV strain (icGDO3-S) was constructed using synthetic biology and reverse genetics to generate a chimeric virus encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV, and represents a strain of SARS-CoV that emerged into the human population independently of the epidemic strain. VRP-S, but not VRP-N, provided protection for both young and senescent mice when challenged with the epidemic strain. When challenged with icGDO3-S, VRP-S protected young mice but only partially protected senescent animals. VRP-N vaccinated mice demonstrated enhanced pulmonary inflammation, which included eosinophils among the cellular infiltrates, following SARS-CoV or icGDO3-S challenge. The highly conserved region at the carboxy-terminus of the coronavirus replicase ORF1a polyprotein is processed by the main proteinase (Mpro) into mature products including nsp7, nsp8, nsp9 and nsp10, proteins with predicted or identified activities involved with RNA synthesis. Mpro continuous translation and processing of ORF1ab polyproteins is required for replication, but specific cleavage events may be dispensable. We determined the requirement for the nsp7-10 proteins and their proteolytic processing during the replication of murine hepatitis virus (MHV), which is phylogenetically grouped with the human coronaviruses OC43 and SARS-CoV. Using the MHV reverse genetics system, in frame deletions of the coding sequences for nsp7, 8, 9, and 10 were either deleted, or the flanking cleavage sites ablated, and the effect upon replication determined. Viable viruses were characterized through analysis of Mpro processing, subgenomic RNA transcription, and in vitro growth fitness. Deletion of any of the four regions encoding nsp7 through 10 was lethal. Disruption of the cleavage sites flanking the protein domains were lethal with the exception of the nsp9/10 cleavage site, which resulted in a mutant virus with severely attenuated replication. In order to determine if a distinct function could be attributed to preprocessed forms of the replicase polyprotein including nsp7-10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus MHV8/7 was not viable, suggesting that the noncleaved intermediate protein may be essential for replication or proteolytic processing

On the Effectiveness of Rebuilding RNA Secondary Structures from Sequence Chunks

Despite the computing power of emerging technolo-gies, predicting long RNA secondary structures with thermodynamics-based methods is still infeasible, espe-cially if the structures include complex motifs such as pseu-doknots. This paper presents preliminary results on rebuilding RNA secondary structures by an extensive and systematic sampling of nucleotide chunks. The rebuilding approach merges the significant motifs found in the secondary struc-tures of the single chunks. The extensive sampling and pre-diction of nucleotide chunks are supported by grid tech-nology as part of the RNAVLab functionality. Significant motifs are identified in the chunk secondary structures and merged in a single structure based on their recurrences an

Supply Chain Resilience in a Pandemic: The Need for Revised Contingency Planning

Organizations have worked over the years to develop efficiencies to their supply chains, which includes efforts to reduce waste, lower costs, consolidate suppliers and distributors, better manage costs of goods sold and inventory, develop efficiencies in packaging, storage, and shipping of product, as well as utilizing digital analytics to manage consumer choices and demands. These are all by-products of world-class manufacturing which have promoted systematic organizational and supply chain efficiencies. However, under economic shocks that are sustained over longer periods of time (e.g., Covid-19 Pandemic) and that affect supply chains from a variety of disruptions, a supply chain that is not prepared or adaptable may be broken or at a minimum weigh down the organization. Therefore, the ability to manage and control risk is a key aspect of effective supply chain management. However, the literature on pandemic risk mitigation is nascent. Thus, this paper offers a review of the extant literature, provides a strategic mitigation model covering five dimensions: leadership, preparedness, digitalization, resilience, and pivoting. These dimensions are designed to help organizations in the future to be more adaptive to events such as global pandemics and other large-scale disruptions and discuss implications for future research

A Meeting of Minds: In Recognition of the Contributions of Randall J. Cohrs

A Special Issue in memory of Randall J. Cohrs, Ph.D. Topics include original research reports on a variety of viruses as well as reviews and commentaries on Randy’s contributions to many investigations

Asymptotic results in-over and under-representation of words in DNA

Master'sMASTER OF SCIENC

Computational Methods for the Analysis of Genomic Data and Biological Processes

In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality