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- ItemImpact of the structural integrity of the three-way junction of adenovirus VAI RNA on PKR inhibition(Public Library of Science, 2017) Dzananovic, Edis; Astha; Chojnowski, Grzegorz; Deo, Soumya; Booy, Evan P.; Padilla-Meier, Pauline; McEleney, Kevin; Bujnicki, Janusz M.; Patel, Trushar R.; McKenna, Sean A.Highly structured RNA derived from viral genomes is a key cellular indicator of viral infection. In response, cells produce the interferon inducible RNA-dependent protein kinase (PKR) that, when bound to viral dsRNA, phosphorylates eukaryotic initiation factor 2αand attenuates viral protein translation. Adenovirus can evade this line of defence through transcription of a non-coding RNA, VAI, an inhibitor of PKR. VAI consists of three base-paired regions that meet at a three-way junction; an apical stem responsible for the interaction with PKR, a central stem required for inhibition, and a terminal stem. Recent studies have highlighted the potential importance of the tertiary structure of the three-way junction to PKR inhibition by enabling interaction between regions of the central and terminal stems. To further investigate the role of the three-way junction, we characterized the binding affinity and inhibitory potential of central stem mutants designed to introduce subtle alterations. These results were then correlated with small-angle X-ray scattering solution studies and computational tertiary structural models. Our results demonstrate that while mutations to the central stem have no observable effect on binding affinity to PKR, mutations that appear to disrupt the structure of the three-way junction prevent inhibition of PKR. Therefore, we propose that instead of simply sequestering PKR, a specific structural conformation of the PKR-VAI complex may be required for inhibition.
- ItemNanoscale structure determination of Murray Valley encephalitis and Powassan virus non-coding RNAs(MDPI, 2020) Mrozowich, Tyler; Henrickson, Amy; Demeler, Borries; Patel, Trushar R.Viral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5’ and 3’ non-coding terminal regions are critical for their survival. Information on their structural features is essential to gain detailed insights into their functions and interactions with host proteins. In this study, the 5’ and 3’ terminal regions of Murray Valley encephalitis virus and Powassan virus were examined using biophysical and computational modeling methods. First, we used size exclusion chromatography and analytical ultracentrifuge methods to investigate the purity of in-vitro transcribed RNAs. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs,which suggest that the 3’ terminal regions are highly extended as compared to the 5’ terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to reinforce that the 5’ terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3’ terminal regions.
- ItemHuman DDX17 unwinds Rift Valley fever virus non-coding RNAs(MDPI, 2020) Nelson, Corey R.; Mrozowich, Tyler; Park, Sean M.; D'Souza, Simmone; Henrickson, Amy; Vigar, Justin R. J.; Wieden, Hans-Joachim; Owens, Raymond J.; Demeler, Borries; Patel, Trushar R.Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135–555 interacting with and unwinding RVFV non-coding regions
- ItemBiodefense implications of new-world hantaviruses(Frontiers Media, 2020) D'Souza, Michael H.; Patel, Trushar R.Hantaviruses, part of the Bunyaviridae family, are a genus of negative-sense, single-stranded RNA viruses that cause two major diseases: New-World Hantavirus Cardiopulmonary Syndrome and Old-World Hemorrhagic Fever with Renal Syndrome. Hantaviruses generally are found worldwide with each disease corresponding to their respective hemispheres. New-World Hantaviruses spread by specific rodent-host reservoirs and are categorized as emerging viruses that pose a threat to global health and security due to their high mortality rate and ease of transmission. Incidentally, reports of Hantavirus categorization as a bioweapon are often contradicted as both US National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention refer to them as Category A and C bioagents respectively, each retaining qualitative levels of importance and severity. Concerns of Hantavirus being engineered into a novel bioagent has been thwarted by Hantaviruses being difficult to culture, isolate, and purify limiting its ability to be weaponized. However, the natural properties of Hantaviruses pose a threat that can be exploited by conventional and unconventional forces. This review seeks to clarify the categorization of Hantaviruses as a bioweapon, whilst defining the practicality of employing New-World Hantaviruses and their effect on armies, infrastructure, and civilian targets.
- ItemMolecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replilcase expression(American Society for Biochemistry and Molecular Biology, 2020) Patel, Ankoor; Treffers, Emmely E.; Meier, Markus; Patel, Trushar R.; Stetefeld, Jörg; Snijder, Eric J.; Mark, Brian L.Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.
- ItemHost transcription factors in hepatitis B virus RNA synthesis(MDPI, 2020) Turton, Kristi L.; Meier-Stephenson, Vanessa; Badmalia, Maulik D.; Coffin, Carla S.; Patel, Trushar R.The hepatitis B virus (HBV) chronically infects over 250 million people worldwide and is one of the leading causes of liver cancer and hepatocellular carcinoma. HBV persistence is due in part to the highly stable HBV minichromosome or HBV covalently closed circular DNA (cccDNA) that resides in the nucleus. As HBV replication requires the help of host transcription factors to replicate, focusing on host protein–HBV genome interactions may reveal insights into new drug targets against cccDNA. The structural details on such complexes, however, remain poorly defined. In this review, the current literature regarding host transcription factors’ interactions with HBV cccDNA is discussed.
- ItemMolecular mechanisms of viral hepatitis induced hepatocellular carcinoma(Baishideng Publishing, 2020) D'Souza, Simmone; Lau, Keith C. K.; Coffin, Carla S.; Patel, Trushar R.Chronic infection with viral hepatitis affects half a billion individuals worldwide and can lead to cirrhosis, cancer, and liver failure. Liver cancer is the third leading cause of cancer-associated mortality, of which hepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. Solid tumors like HCC are complex and have heterogeneous tumor genomic profiles contributing to complexity in diagnosis and management. Chronic infection with hepatitis B virus (HBV), hepatitis delta virus (HDV), and hepatitis C virus (HCV) are the greatest etiological risk factors for HCC. Due to the significant role of chronic viral infection in HCC development, it is important to investigate direct (viral associated) and indirect (immune-associated) mechanisms involved in the pathogenesis of HCC. Common mechanisms used by HBV, HCV, and HDV that drive hepatocarcinogenesis include persistent liver inflammation with an impaired antiviral immune response, immune and viral protein-mediated oxidative stress, and deregulation of cellular signaling pathways by viral proteins. DNA integration to promote genome instability is a feature of HBV infection, and metabolic reprogramming leading to steatosis is driven by HCV infection. The current review aims to provide a brief overview of HBV, HCV and HDV molecular biology, and highlight specific viral-associated oncogenic mechanisms and common molecular pathways deregulated in HCC, and current as well as emerging treatments for HCC.
- ItemCurrent approaches for RNA-labelling to identify RNA-binding proteins(Canadian Science Publishing, 2020) Gemmill, Darren; D'souza, Simmone; Meier-Stephenson, Vanessa; Patel, Trushar R.RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA–protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the “bait” and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.
- ItemStructural and hydrodynamic characterization of dimeric human oligoadenylate synthetase 2(2020) Koul, Amit; Gemmill, Darren; Lubna, Nikhat; Meier, Markus; Krahn, Natalie; Booy, Evan P.; Stetefeld, Jörg; Patel, Trushar R.; McKenna, Sean A.Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.
- ItemMolecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox(Elsevier, 2021) Rutbeek, Nicole R.; Rezasoltani, Hanieh; Patel, Trushar R.; Khajehpour, Mazdak; Prehna, GerdStreptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and a pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as prophages. These genetic elements encode both the phage proteins and the toxins harmful to the human host. One such conserved phage protein, paratox (Prx), is always found encoded adjacent to the toxin genes, and this linkage is preserved during all stages of the phage life cycle. Within S. pyogenes, Prx functions to inhibit the quorum-sensing receptor-signal pair ComRS, the master regulator of natural competence, or the ability to uptake endogenous DNA. However, the mechanism by which Prx directly binds and inhibits the receptor ComR is unknown. To understand how Prx inhibits ComR at the molecular level, we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access its DNA-binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the interdomain conformational change required for activation of ComR via the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing and highlights the versatility of small phage proteins.
- ItemHuman DDX3X unwinds Japanese encephalitis and Zika viral 5' terminal regions(MDPI, 2021) Nelson, Corey R.; Mrozowich, Tyler; Gemmill, Darren L.; Park, Sean M.; Patel, Trushar R.Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5′ terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5′ TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5′ TR of JEV and demonstrated its direct interaction with recombinant DDX3X (Kd of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5′ and 3′ TRs of flaviviruses, we investigated if the ZIKV 5′ TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5′ TR with a Kd of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5′ TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication
- ItemIdentification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA(Elsevier, 2021) Meier-Stephenson, Vanessa; Badmalia, Maulik D.; Mrozowich, Tyler; Lau, Keith C. K.; Schultz, Sarah K.; Gemmill, Darren L.; Osiowy, Carla; van Marle, Guido; Coffin, Carla S.; Patel, Trushar R.Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.
- ItemBioinformatic analysis of structure and function of LIM domains of human zyxin family proteins(MDPI, 2021) Siddiqui, M. Quadir; Badmalia, Maulik D.; Patel, Trushar R.Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and functions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share similarities with transcriptional regulators and have positively charged electrostatic patches, which may indicate that they have previously unanticipated nucleic acid binding properties. Intrinsic dynamics analysis of Lim domains suggest that only Lim1 has similar internal dynamics properties, unlike Lim2/3. Furthermore, we analyzed protein expression and mutational frequency in various malignancies, as well as mapped protein-protein interaction networks they are involved in. Overall, our comprehensive bioinformatic analysis suggests that these proteins may play important roles in mediating protein-protein and protein-nucleic acid interactions.
- ItemPurification and characterization of inorganic pyrophosphatase for in vitro RNA transcription(Canadian Science Publishing, 2022) Tersteeg, Scott; Mrozowich, Tyler; Henrickson, Amy; Demeler, Borries; Patel, Trushar R.Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precip- itating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.