BMBDG Seminar: Ph.D. Exit Seminar – Jibin Sadasivan and Reid Warsaba
Jibin Sadasivan
Title: When proteins go viral: Investigation on how a viral protein impairs stress granule formation
Abstract: Stress granules (SG) are ribonucleoprotein aggregates that accumulate during cellular stress when translation is limited. Inhibition of SG assembly has been observed under virus infection across species, suggesting a conserved fundamental viral strategy. How this occurs and why this would benefit virus infection are not fully understood. The 1A protein encoded by the model dicistrovirus, cricket paralysis virus (CrPV), is a multifunctional viral protein that can inhibit SG formation and bind to and degrade Argonaute-2 (Ago-2) in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway. Moreover, the R146 residue at the C terminus of 1A is necessary for virus infection in Drosophila S2 cells and flies. Here, we uncouple CrPV-1A’s functions and provide insights into its underlying mechanism for SG inhibition. CrPV-1A’s ability to inhibit SG formation does not require the Ago-2 binding domain but does require the E3 ubiquitin ligase binding domain. Overexpression and infection studies in Drosophila and human cells showed that wild-type CrPV-1A but not mutant R146A CrPV-1A localizes to the nuclear membrane, which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome analysis demonstrated that a single R146A mutation dramatically dampens host transcriptome changes in CrPV-infected cells. Finally, inhibition of SG formation by CrPV-1A requires Ranbp2/Nup358 in an R146-dependent manner. Wse propose that CrPV utilizes a multifaceted strategy for productive virus infection whereby the CrPV-1A protein interferes with a nuclear event that contributes to the suppression of SG assembly.
Reid Warsaba
Title: Multiple Viral Protein Genome Linked Proteins in Dicistrovirus Infection
Abstract: Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of positive-sense single-stranded RNA viruses. VPg is covalently linked to the 5’ end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses encode multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use the Dicistrovirus, Cricket paralysis virus (CrPV), which encodes four non-identical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses encode two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1-4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, from which mutations in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant-negative effect. Using viral minigenome reporters that monitor Dicistrovirus 5’ untranslated region (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of IGR IRES:5’ UTR IRES translation. We uncover a novel viral strategy whereby VPg copies in Dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote virus infection. We also performed a bioinformatic analysis of Dicistrovirus VPgs and find many novel Dicistroviruses with repeated VPgs, up to eight copies and find that the VPg type but not the number correlates with the RdRp evolution of Dicistroviruses.
Monday, October 03, 2022 at 2:30 pm – 3:30 pm at LSC 3 and Zoom
Hosted by: Dr. Eric Jan
2022/23 BMB Mentorship Program
The Biochemistry and Molecular Biology (BMB) mentorship program matches mentors and mentees within BMB to build meaningful connections with a professional, career, and scientific focus. This initiative aims to foster a sense of community within the BMB program with an emphasis on Equity, Inclusion, and Diversity (EDI).
This program is open to all members of the BMB department (Undergrads, Grad students, postdocs, PI’s) and will be running from September 2022 – April 2023. This initiative aims to foster a sense of community within the BMB program with an emphasis on Equity, Diversity, and Inclusion.
The sign-up deadline for the BMB Mentorship Program is this Friday, September 23.
BMBDG Seminar: Dr. Tom Hobman
Title: Unraveling RNA virus-host interactions reveals novel antiviral targets.
Professor. Department of Cell Biology.
University of Alberta
Abstract: RNA virus infections impose huge economic and social burdens around the globe. Direct-acting antiviral therapeutics and vaccines can be highly effective in controlling epidemic and pandemic viruses, but these drugs/prophylactics take time to develop and their efficacy is often limited to specific viral strains. To minimize the impact of future emerging RNA viruses, a stockpile of broad-spectrum antivirals is required to serve as a first line of defense against these pathogens. Identification of host cell-based pathways that are exploited or inhibited by multiple viruses is expected to reveal novel targets for antiviral therapy. Using targeted and unbiased screening approaches, we have identified multiple host cell pathways that can be pharmacologically inhibited or enhanced to reduce infection by multiple RNA viruses including SARS-CoV-2..
Monday, September 26, 2022 at 2:30 pm at LSC 3 and Zoom
Hosted by: Dr. Eric Jan
New Faculty Announcement – Dr. Eden Fussner-Dupas
The Department of Biochemistry & Molecular Biology is pleased to announce the appointment of Dr. Eden Fussner-Dupas as Assistant Professor of Teaching! Dr. Fussner-Dupas will be teaching BIOC 302 this fall.
Dr. Fussner-Dupas’ profile page: https://biochem.ubc.ca/person/eden-fussner-dupas/
BMBDG Seminar: P.h.D Exit Seminar – Amy Strilchuk (Cancelled)
Title: Blood clot stability can be controlled using lipid nanoparticle-delivered siRNA
Abstract: Disruptions in the balance of clot formation and degradation can lead to dangerous, potentially fatal, clotting or bleeding events. While a plethora of research tools and clinical therapies exist to control clot formation, options for controlling clot degradation are extremely limited. RNA and lipid-based technologies can be applied to the field of hematology and meet the need for long-acting agents to control the stability of clots. This work developed siRNA-LNP agents that increase or decrease the durability of clots in three model species, enabling further investigation of this pathway as a target for research in a wide range of coagulation disorders.
Monday, September 19, 2022 at 2:30 pm – 3:30 pm via Zoom
Hosted by: Dr. Christian Kastrup
BMBDG Seminar: Dr. Roberto Chica
Title: Ensemble-based computational design of enzyme catalysis and conformational equilibrium.
Professor, Department of Chemistry and Biomolecular Sciences.
University of Ottawa
Abstract: Enzymes are dynamic molecules, and this flexibility is essential to their catalytic function. Yet, computational enzyme design is typically performed using a single protein scaffold as design template, ignoring the important contributions of dynamics in enzyme catalysis. In the past few years, my group has developed multistate computational protein design methods that allow proteins to be modelled as structural ensembles that more realistically represent the range of conformations that these molecules can adopt. Here, I will show how we can use structural ensembles to accurately design more efficient de novo enzymes than previously possible and remodel an enzyme conformational landscape to obtain an 1800-fold substrate selectivity switch.
Monday, September 12, 2022 at 2:30 pm at LSC 3 and Zoom
Hosted by: Dr. Nobuhiko Tokuriki
BMBDG Seminar: Ph.D. Exit Seminar – Karlton Scheu
Title: Dissecting the biophysical properties and DNA-binding specificity of the ETV6 transcription factor
Abstract: Transcription factors bind to specific DNA sequences and regulate the expression of associated genes. In this thesis, I used several biophysical methods to investigate the structure and DNA-binding specificity profile of the eukaryotic transcription factor ETV6. I investigated the mechanisms by which ETV6 selectively binds its cognate DNA targets. ETV6 prefers DNAs containing a core GGAA motif, unlike other family members where GGA(A/T) is accepted. This specificity toward the fourth adenine is mediated by a single histidine, His396, contrasted with a tyrosine in other family members. Through NMR-monitored pH titrations, I found His396 adopts the neutral Nε2H tautomeric state when ETV6 is both free and DNA-bound. Both structural and surface plasmon resonance binding studies revealed the mutation of His396 to a tyrosine increased its affinity for GGAT-containing DNA, while not diminishing its affinity for DNAs with a GGAA core. Thus I propose that His396 does not serve to enable binding of DNA containing a GGAA core, but rather to disfavour association toward DNAs containing bases other than an adenine at this fourth position. This thereby restricts the transcriptional profile of ETV6 relative to other ETS factors. In a related study I investigated conformational flexibility within the DNA-binding domain of ETV6. All ETS family proteins share a structurally conserved ETS domain, allowing association toward many redundant DNA sequences. These proteins can also recognize subtly different DNA regions to enable transcriptional specificity. I proposed a contributing factor toward the profile of DNAs each family member recognized was domain flexibility. Testing this, I introduced cavity-forming mutations in the ETV6 ETS domain, resulting in increase flexibility determined through relaxation-dispersion NMR experiments. Then, using microarrays, I showed the increase in flexibility weakened selection of near-cognate DNAs, and thereby increased the specificity for cognate DNAs. These flexibility changes predominantly affected selection of DNA bases contacted solely via their phosphodiester backbone, indicating a link between protein flexibility and specificity for the sequence-dependent shape of DNA. Collectively my thesis uncovered many biophysical properties of the ETV6 ETS domain and illustrated how these features contribute to its DNA binding specificity at a molecular level.
Monday, August 29, 2022 at 2:30 pm – 3:30 pm via Zoom
Hosted by: Dr. Lawrence McIntosh