1,721,008 research outputs found
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Investigating the Role of the Zinc-Finger PARPs in Mediating Antiviral Innate Immunity
Get PDFMany viruses seek to manipulate the cellular environment during infection by seizing control over host post translational modifications (PTMs) by using viral E3-ubiquitin ligases, phosphatases, and kinases. Some viruses, including the genus of Alphaviruses, encode for a macrodomain, an enzymatic protein domain capable of reversing the PTM of ADP-ribosylation (ADPr). ADPr is found in a diverse range of host processes ranging from DNA damage and repair to protein quality control. When their macrodomains are disrupted, viruses lose infectious capacity, drawing attention to the importance of this modification. Identifying which host proteins are involved with this interaction could be key to understanding how these viruses are functioning and how they are manipulating the cell. The host factors at play are known as PARPs, which stands for poly-ADP-ribose-polymerases. Here, I have explored a number of these host PARPs and found novel ways in which they interact with alphaviruses to restrict viral replication. A number of PARPs are implicated in the interaction, most specifically those that either bear a macrodomain themselves, the macroPARPs of PARP9,14,and 15, or the Zinc Finger PARPS, PARP7,12, and 13. Further exploration of these PARPs, how they interact with both viral proteins and each other, is critical to fully understanding the role that ADP-ribosylation plays in restricting Alphavirus infection, as well as how alphaviruses are subverting that control
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Evolutionary Warfare: Characterizing Viral Antagonism of Host Innate Immune Components
Get PDFHost-pathogen conflicts are major drivers of evolution and generate a seemingly endless array of challenges to the fields of medicine and scientific research. While extensive work has been conducted to combat diseases caused by viruses and to understand their molecular mechanisms, there is a need for better tools to identify and predict host components that have interacted with viruses over evolutionary history. By seeking out these evolutionary battlegrounds, we can discover novel and otherwise difficult-to-identify struggles between viruses and their hosts. Many logical host targets, such as sensors of non-self markers and effectors of the immune system have been characterized as important players in host defense. Here, I describe two instances of the evolution-guided discovery of unique viral interactors of the host innate immune response. The first chapter characterizes a host-evolved “tripwire” immune sensor that baits viruses into activation, and the second chapter details virus-driven evolution of a previously undescribed effector of the host innate immune response
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A Comparative Transcriptomic Analysis of the Mosquito Blood Feeding Response
Get PDFFemale mosquitoes are only able to produce eggs upon feeding on blood from their vertebrate hosts, but this feeding behavior activates a network of genes to ensure their survival following a blood meal. Blood meals are critical to mosquito proliferation despite activating stress and defense responses, however the whole transcriptome at the early time point post-blood meal has not been fully studied across mosquito genera. To explore the transcriptomic responses under environmental thermal stress and early-stage blood feeding, we compared bulk RNA-seq profiles across Ae. aegypti, Ae. albopictus, An. gambiae, An. stephensi, and Cx. quinquefasciatus under these experimental conditions. In addition, we conducted a functional and orthology inference analysis to compare differentially expressed genes across species and condition. We found that despite the conservation of the heat shock response, there are deviations in the response across species based on heat shock inducibility. We were also able to identify that a blood meal elicits a heat shock response in Ae. albopictus and that intersections with the blood feeding response in Ae. albopictus highlights the temporal dynamics to survival thermal stress of a blood meal before the activation of metabolic processes. Our comparative approach and generated RNA-seq datasets illustrates the convergence and divergence in these critical stress responses of mosquitoes that would be beneficial for understanding mosquito survival
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NLRP10 is cleaved by diverse flavivirus proteases and is a potent regulator of inflammasome activation
Get PDFFlaviviruses such as dengue, Zika, West Nile, and yellow fever are dangerous viruses that pose a significant global health burden and have massive epidemic potential. Annually, these viruses infect over 400 million individuals worldwide and have been responsible for several pandemics in recent years. Flavivirus infection can result in hepatitis, microcephaly, liver cirrhosis, hemorrhagic shock, and even death. Despite these severe consequences, there are no available therapeutics for the majority of these flaviviruses . The initial response to flavivirus infection is characterized by the mass production of pro-inflammatory molecules in the host’s attempt to defend against pathogen invasion, causing widespread tissue damage through a phenomenon known as a cytokine storm. Consequently, it is crucial to investigate the host immune regulators that govern inflammatory multi-protein complexes called inflammasomes. Previous studies have highlighted the role of Nod-Like Receptor Protein 10 (NLRP10) in suppressing inflammation by inhibiting another human protein called pro-caspase-1 (pro-CASP1), a key component of many inflammasome systems.
Therefore, we sought to determine what role NLRP10 played in the context of flavivirus-associated inflammation. Our findings reveal that NLRP10 is cleaved by diverse flavivirus proteases at amino acid site 265. However, this cleavage did not result in the restoration of inflammasome activation. Despite this, we also discovered that flaviviruses also independently antagonize inflammasomes. Collectively, our results demonstrate that flaviviruses exert evolutionary pressures on many inflammasome-associated proteins such as NLRP10, highlighting the intricacy of virus-host dynamics on a broader pan-flavivirus scale
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Evolution-guided Discovery of Species-specific Viral Protease Targets
Get PDFMany pathogens encode proteases that serve to antagonize the host immune system. In particular, viruses with a positive-sense single-stranded RNA genome ((+)ssRNA), including picornaviruses, flaviviruses, and coronaviruses, encode proteases that are not only required for processing the viral polyprotein into functional units but also manipulate crucial host cellular processes through their proteolytic activity. Because these proteases must coordinate both the cleavage of numerous polyprotein sites and subversion of host immunity, evolution of viral proteases is expected to be highly constrained. Despite this strong evolutionary constraint, these viral proteins and host immune factors are engaged in “evolutionary arms races” that results in diverse protease-host interactions even within closely related species. In some cases, rapid host gene evolution can result in avoidance of cleavage by viral proteases. In other, more recently described cases, hosts can evolve to bait viral proteases into cleaving them using a “tripwire” strategy of immune activation. Such data provide an explanation for why viral polyprotein sites evolve despite such a strong evolutionary constraint and highlight the importance of identifying and characterizing host proteins that are targeted by viral proteases. Moreover, such an evolutionary model provides insight into the changes in molecular functions between viral proteases and host factors, and underscores the role of viral proteases in viral host range, zoonosis and host immune gene evolution. Here, I describe a combined computational and functional approach that guide the discovery of new host targets of viral proteases, including the characterization of two new host innate immunity tripwires that trigger inflammation in response to viral protease activity
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PARP13 Is Required for IFN-γ-Mediated Inhibition of Sindbis Virus
Get PDFIntrinsic antiviral immunity is a critical mediator against viral infection. It is composed of both constitutively expressed antiviral restriction factors and others that are upregulated during infection. Research over the past several years has shown that while type-I and -III IFNs have been traditionally thought to be the primary drivers of the intrinsic antiviral immune response, type-II IFN has clear antiviral capabilities as well - although many of the details remain shrouded in mystery. Our lab recently showed that PARP13, a robust restriction factor capable of attenuating a wide variety of viruses, has two isoforms with distinctly different functions in type-I IFN signaling. Here, we show that PARP13L, but not PARP13S, is required for IFN-γ-mediated inhibition of Sindbis Virus, and potentially other alphaviruses as well. We additionally show that PARP13L is not upregulated by IFN-γ and that other members of the PARP family are involved, and lastly describe future directions for the preliminary data shown here
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Enterovirus 3C Cleavage of RIPK3 Dysregulates Inflammatory Signaling and Drives Rapid Evolution in Primates
No full textDespite being a critical component of the antiviral response, inflammatory signaling pathways are known to be hijacked by pathogens from diverse viral families, resulting in poor health outcomes for infected hosts. Receptor interacting protein kinase 3 (RIPK3) is a key inflammatory mediator traditionally studied for its role facilitating apoptosis and necroptosis, a highly inflammatory and lytic cell death pathway. During viral infection, RIPK3 dysregulation contributes to increased virus replication. In fact, enterovirus-encoded 3C proteases are known to cleave RIPK3 to disrupt activation of necroptosis. However, in addition to mediating cell death, RIPK3 is also associated with cell survival and activation of the nuclear factor kappa B (NF-κB) transcription factor; Upon phospho-activation in the cytoplasm, the NF-κB complex translocates to the nucleus, stimulating the production of pro-inflammatory cytokines. Whilst viruses are known to dysregulate inflammatory cell death signaling from RIPK3, the implications of cleavage on NF-κB activation are extremely understudied. Moreover, RIPK3 is known to be undergoing rapid evolution in primates, with over 20 residues showing significant rates of mutation throughout the protein, including some in close proximity to the enterovirus cut site. Using an in vitro bioluminescent reporter assay, we show that the enterovirus 3C protease will reduce NF-κB activation through cleavage of RIPK3 when overexpressed in HEK293T cells. Moreover, we show that primate amino acid residues at and around the enterovirus cut site confer protection from viral cleavage of RIPK3 and restores NF-κB activity. Finally, we demonstrate that viral antagonism of RIPK3 orthologues from a diverse array of vertebrate species is well conserved. The results of our study demonstrate the importance of inhibiting RIPK3-induced inflammatory signaling for enterovirus pathogenesis, and the protective diversity in RIPK3 sequence seen in primates suggests a host-virus arms race interaction between RIPK3 and enteroviruses, explaining the rapid evolution observed with this protein
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Molecular Mechanisms that Underpin Antigen Receptor Recombination: Exploring the Role of E2A and BRD4 on V(D)J Recombination in B Cells
Get PDFA diverse antibody repertoire is the backbone of a successful adaptive immune response. This diversity is created in developing B cells by the rearrangement of the V (variable), D (diversity), and J (joining) gene segments, termed V(D)J recombination, to produce unique antigen specific B cell receptors (BCRs). V(D)J recombination is regulated by an ensemble of transcription factors, co- activators as well as architectural proteins. We know that certain V gene regions are more highly rearranged in wild type cells, yet the mechanism for this bias is not well known. We propose that the enhancer-associated E2A, p300 and BRD4 proteins influence the frequency and pattern of gene rearrangement. Using CRISPR/Cas9 technology, we have successfully deleted E2A as well as BRD4 to determine the effects of each mutation on the frequency and pattern of rearrangement in the immunoglobulin kappa locus (Igκ) locus of an inducible cell line. We have examined the consequences of these deletions on Vκ rearrangements. Notably, our results indicate that E2A and BRD4 are responsible for influencing the pattern and frequency of Vκ gene rearrangement. These data indicate that E2A and BRD4 are key factors in modulating nuclear architecture and the Igκ repertoire
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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