Z-DNA Binding Protein 1 (ZBP1)-Mediated Cell Death During Infection by Diverse RNA Viruses is Dependent on the Zα2 Domain

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Description
Z-DNA binding protein 1 (ZBP1) is an interferon-inducible protein that plays a crucial role in antiviral defense by recognizing Z-form nucleic acid (Z-NA), a left-handed conformer of double-stranded DNA/RNA. When ZBP1 binds to Z-NA, it can trigger programmed cell death…

Z-DNA binding protein 1 (ZBP1) is an interferon-inducible protein that plays a crucial role in antiviral defense by recognizing Z-form nucleic acid (Z-NA), a left-handed conformer of double-stranded DNA/RNA. When ZBP1 binds to Z-NA, it can trigger programmed cell death pathways, including apoptosis and necroptosis, in collaboration with receptor interacting protein kinases 1 and 3 (RIPK1 and RIPK3). Z-NA positive viruses including poxviruses and influenza A virus (IAV) activate ZBP1-dependent cell death during replication. Little is known whether ZBP1 plays any role during Z-NA negative virus infection. Doxycycline-inducible A549 ACE2 Tet-On cells were constructed to express ZBP1 and were infected with Z-NA negative viruses. ZBP1-expressing cells infected with Sindbis virus (SINV), La Crosse virus (LACV), Vesicular stomatitis virus (VSV) and human coronavirus OC43 (hCoV-OC43) underwent extensive cell death, which could be rescued by a caspase inhibitor but not by JAK1/2 or RIPK1 kinase inhibitors. However, cell death was not observed upon Zika virus (ZIKV), Encephalomyocarditis virus (EMCV), Chikungunya virus (CHKV) or human coronavirus 229E (hCoV-229E) infection. ZBP1 expression did not impact the replication of all tested viruses. In addition, ZBP1-mediated cell death during infection depends on the Zα2 and RHIM1 domains and partially on the C-terminal domain. These findings suggest that Z-NA can be detected by the Zα2 domain to initiate cell death pathways during infection with some Z-NA negative viruses and that the RHIM1/C-terminal domains are necessary for ZBP1-induced cell death. Further research is needed to determine the Z-NA ligand and the precise mechanism of ZBP1-mediated antiviral responses and how they can be exploited for the development of novel antiviral therapies.
Date Created
2023
Agent

A Multiplexed Lateral Flow Assay for The Serologic Point of Care Detection of HPV Positive Cervical Cancer

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Description
Human Papillomavirus (HPV) is the most commonly transmitted STI and isresponsible for an estimated 5% of cancer cases worldwide. HPV infection is implicated in 70% of cervical cancer incidence and is also responsible for a variety of oropharyngeal and anogenital cancers. While…

Human Papillomavirus (HPV) is the most commonly transmitted STI and isresponsible for an estimated 5% of cancer cases worldwide. HPV infection is implicated in 70% of cervical cancer incidence and is also responsible for a variety of oropharyngeal and anogenital cancers. While vaccination has greatly reduced the cervical cancer burden in developed countries, HPV infection remains high in developing countries due to high cost and poor access to healthcare. Several studies have highlighted the presence of anti-HPV antibodies following infection and their potential use as biomarkers for developing novel screening methods. Progression from initial infection to cancer is slow, thus presenting an opportunity for effective screening programs. Biomarker screening is an important area of cancer detection and Lateral Flow Assays (LFA) are a low cost, easy to use alternative to other screening methods that require extensive training and laboratory space. Therefore, this project proposes as a hypothesis that the development of an LFA screening for HPV specific IgG can provide clinically relevant data for the early detection of cervical dysplasia. This project adapts an LFA in a multiplexed format for fluorescence-based serologic detection of HPV specific IgG in patient plasma.
Date Created
2023
Agent

The Mechanisms of Alpha Herpesvirus Intracellular Trafficking and Exocytosis

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Description
Alpha herpesviruses are a family of neuroinvasive viruses that infect multiplevertebrate species. Alpha herpesviruses are responsible for human and livestock infections, most notably Herpes Simplex Virus (HSV), Varicella Zoster virus (VZV), and Pseudorabies Virus (PRV). PRV is a potent swine…

Alpha herpesviruses are a family of neuroinvasive viruses that infect multiplevertebrate species. Alpha herpesviruses are responsible for human and livestock infections, most notably Herpes Simplex Virus (HSV), Varicella Zoster virus (VZV), and Pseudorabies Virus (PRV). PRV is a potent swine virus that can infect other mammals, and results in lethal encephalitis that can be devastating to livestock and of great financial expense to farmers. HSV, types 1 and 2, and VZV are widespread throughout the global human population, with estimates of the HSV-1 burden at about 60% of people worldwide. The hallmark of alpha herpesvirus infection is a persistent, lifelong infection that can reactivate throughout the lifespan of the host. Currently, the precise mechanisms of how these viruses undergo intracellular trafficking to emerge from the infected cell in epithelial tissues is not well understood. Many insights have been made with PRV in animal neurons, both in culture systems and animal models, about the viral genes and host factors involved in these processes. However, understanding of these mechanisms, and the interplay between viral and host proteins, in the human pathogen HSV-1 is even more lacking. Using recombinant fluorescent virus strains of HSV-1 and Total Internal Reflection Microscopy to image the transport of mature viral progeny in epithelial cells, it was determined that the egress of HSV-1 uses constitutive cellular secretory pathways. Specifically, the viral progeny traffic from the trans-Golgi network to the site of exocytosis at the plasma membrane via Rab6a secretory vesicles. This work will contribute to the understanding of how alpha herpesviruses complete their lifecycles in host cells, particularly at the sites where infection initially occurs and can spread to a new organism. Knowledge of these processes may lead to the development of therapeutics or prophylactics to reduce the burden of these viruses.
Date Created
2023
Agent

Serial Crystallographic Studies for Therapeutic Drug Advancement

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Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding…

Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
Date Created
2022
Agent

Using Syringe Pump for Concentrating Particles in Microfluidic Device

Description

An electric field can be applied to a microfluidic device in order to stop particle flow. Electroosmosis, electrophoresis, and dielectrophoresis act on the particles in different directions in the microfluidic channel, and when these forces create zero net force, the…

An electric field can be applied to a microfluidic device in order to stop particle flow. Electroosmosis, electrophoresis, and dielectrophoresis act on the particles in different directions in the microfluidic channel, and when these forces create zero net force, the particle stops in the channel. The goal of the performed experiments is to investigate whether hydrostatic pressure generated by a syringe pump could help concentrate these particles and separate them from other contents. Introducing precise, adjustable hydrostatic pressure from the syringe pump provides another mechanism for controlling particle behavior. A microfluidic channel was crafted into a device connected to a syringe pump, and videos of 1 µm silica particles in the device were recorded under a microscope in order to show that samples could be infused into the device and concentrated or captured at a specific location in the channel using hydrostatic pressure. Capture of the particles occurred with and without controlled hydrostatic pressure, but these events occurred somewhat consistently at different voltages. In addition, particle movement in the channel with the syringe pump off was originally attributed to the electrokinetic forces. However, when compared to experiments without the syringe pump connected to the device, it became evident that the electrokinetic forces should have moved the particles in the opposite direction and that, in actuality, there is an inherent pressure in the device also affecting particle movement even when the syringe pump is not turned on.

Date Created
2022-12
Agent

Vaccinia Virus’ E3 Protein Inhibits Cellular Recognition of Canonical dsRNA and ZRNA

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Description
Poxviruses such as monkeypox virus (MPXV) are emerging zoonotic diseases. Compared to MPXV, Vaccinia virus (VACV) has reduced pathogenicity in humans and can be used as a partially protective vaccine against MPXV. While most orthopoxviruses have E3 protein homologues with…

Poxviruses such as monkeypox virus (MPXV) are emerging zoonotic diseases. Compared to MPXV, Vaccinia virus (VACV) has reduced pathogenicity in humans and can be used as a partially protective vaccine against MPXV. While most orthopoxviruses have E3 protein homologues with highly similar N-termini, the MPXV homologue, F3, has a start codon mutation leading to an N-terminal truncation of 37 amino acids. The VACV protein E3 consists of a dsRNA binding domain in its C-terminus which must be intact for pathogenicity in murine models and replication in cultured cells. The N-terminus of E3 contains a Z-form nucleic acid (ZNA) binding domain and is also required for pathogenicity in murine models. Poxviruses produce RNA transcripts that extend beyond the transcribed gene which can form double-stranded RNA (dsRNA). The innate immune system easily recognizes dsRNA through proteins such as protein kinase R (PKR). After comparing a vaccinia virus with a wild-type E3 protein (VACV WT) to one with an E3 N-terminal truncation of 37 amino acids (VACV E3Δ37N), phenotypic differences appeared in several cell lines. In HeLa cells and certain murine embryonic fibroblasts (MEFs), dsRNA recognition pathways such as PKR become activated during VACV E3Δ37N infections, unlike VACV WT. However, MPXV does not activate PKR in HeLa or MEF cells. Additional investigation determined that MPXV produces less dsRNA than VACV. VACV E3Δ37N was made more similar to MPXV by selecting mutants that produce less dsRNA. By producing less dsRNA, VACV E3Δ37N no longer activated PKR in HeLa or MEF cells, thus restoring the wild-type phenotype. Furthermore, in other cell lines such as L929 (also a murine fibroblast) VACV E3Δ37N, but not VACV WT infection leads to activation of DNA-dependent activator of IFN-regulatory factors (DAI) and induction of necroptotic cell death. The same low dsRNA mutants demonstrate that DAI activation and necroptotic induction is independent of classical dsRNA. Finally, investigations of spread in an animal model and replication in cell lines where both the PKR and DAI pathways are intact determined that inhibition of both pathways is required for VACV E3Δ37N to replicate.
Date Created
2021
Agent

The Potential of Virus-Like Particles (VLPs) as an Effective Vaccine Candidate for the COVID-19 Vaccine: Review

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Description

Virus-like particles (VLPs) are optimum candidates for creating vaccines, as they are highly flexible, adaptable, safe, and similar to the structural proteins of the target cells. The COVID 19 pandemic has increased the need to create effective and safe vaccines…

Virus-like particles (VLPs) are optimum candidates for creating vaccines, as they are highly flexible, adaptable, safe, and similar to the structural proteins of the target cells. The COVID 19 pandemic has increased the need to create effective and safe vaccines that can be mass produced to stop the spread of COVID-19. Till now, various types of vaccine platforms have been utilized to create COVID-19 vaccines, each with unique characteristics and techniques. It is essential to use robust vaccine platforms that can deliver optimum results in a short period of time, with minimal risks. The structural proteins found in SARS-CoV-2, such as Spike (S) protein have been widely targeted to induce antibody response, also called a humoral response, which is a part of acquired immunity. The other structural proteins such as M (membrane) and E (envelope) can also be used as targets for antibodies. The S2 and glycoprotein (S full) can be used to induce an efficient IgG response. Therefore, the incorporation of structural proteins into VLPs can prove to be useful. Furthermore, double mosaic VLPs employs double epitopes, which can effectively cover the distances between the S proteins, thus optimizing the B cell activation process. This review describes the various developments that have taken place in the field of VLPs and more specifically, with regards to developing VLP vaccines against the SARS-CoV-2 virus.

Date Created
2022-05
Agent

Surveillance of Influenza Virus Spread on the Arizona State University Campus

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Description
The 2017-2018 Influenza season was marked by the death of 80,000 Americans: the highest flu-related death toll in a decade. Further, the yearly economic toll to the US healthcare system and society is on the order of tens of billions…

The 2017-2018 Influenza season was marked by the death of 80,000 Americans: the highest flu-related death toll in a decade. Further, the yearly economic toll to the US healthcare system and society is on the order of tens of billions of dollars. It is vital that we gain a better understanding of the dynamics of influenza transmission in order to prevent its spread. Viral DNA sequences examined using bioinformatics methods offer a rich framework with which to monitor the evolution and spread of influenza for public health surveillance. To better understand the influenza epidemic during the severe 2017-2018 season, we established a passive surveillance system at Arizona State University’s Tempe Campus Health Services beginning in January 2018. From this system, nasopharyngeal samples screening positive for influenza were collected. Using these samples, molecular DNA sequences will be generated using a combined multiplex RT-PCR and NGS approach. Phylogenetic analysis will be used to infer the severity and temporal course of the 2017-2018 influenza outbreak on campus as well as the 2018-2019 flu season. Through this surveillance system, we will gain knowledge of the dynamics of influenza spread in a university setting and will use this information to inform public health strategies.
Date Created
2019-05
Agent

Merging single-shot XFEL diffraction data from inorganic nanoparticles: a new approach to size and orientation determination

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Description
X-ray free-electron lasers (XFELs) provide new opportunities for structure determination of biomolecules, viruses and nanomaterials. With unprecedented peak brilliance and ultra-short pulse duration, XFELs can tolerate higher X-ray doses by exploiting the femtosecond-scale exposure time, and can thus go beyond…

X-ray free-electron lasers (XFELs) provide new opportunities for structure determination of biomolecules, viruses and nanomaterials. With unprecedented peak brilliance and ultra-short pulse duration, XFELs can tolerate higher X-ray doses by exploiting the femtosecond-scale exposure time, and can thus go beyond the resolution limits achieved with conventional X-ray diffraction imaging techniques. Using XFELs, it is possible to collect scattering information from single particles at high resolution, however particle heterogeneity and unknown orientations complicate data merging in three-dimensional space. Using the Linac Coherent Light Source (LCLS), synthetic inorganic nanocrystals with a core–shell architecture were used as a model system for proof-of-principle coherent diffractive single-particle imaging experiments. To deal with the heterogeneity of the core–shell particles, new computational methods have been developed to extract the particle size and orientation from the scattering data to assist data merging. The size distribution agrees with that obtained by electron microscopy and the merged data support a model with a core–shell architecture.
Date Created
2017-08-27
Agent

Optimization of a viral system to produce vaccines and other biopharmaceuticals in plants

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Description
Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially…

Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities.

In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
Date Created
2017
Agent
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