Biology subjects

Brindley, M. A.

Publications and source records attributed to Brindley, M. A..

12 recordsLinked to original sources

bioRxiv2024

Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of Chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce Chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process that appears to be mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. ImportanceChikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV egress, to better understand CHIKVs ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKVs ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.

microbiology↗

bioRxiv2023

Fusogenic Structural Changes in Arenavirus Glycoproteins are Associated with Viroporin Activity

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of "leaky" LASV fusion using single pseudoparticle tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small- molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm. Authors summaryFusion of enveloped virus with host cell membranes is mediated by extensive conformational changes in viral glycoproteins, triggered by binding to cognate receptors and/or by exposure to acidic pH in the maturing endosome. We have previously reported that, unlike many other viral glycoproteins, pH-triggered endosomal fusion by the Lassa virus viral glycoprotein complex (GPc) is preceded by a mild permeabilization of the viral membrane. Here, we provide evidence that this activity is associated with early fusogenic changes in the GPc and is markedly enhanced by virus-cell contact. Permeabilization is induced by the glycoproteins of diverse arenaviruses and occurs in multiple target cell types. Based on these observations, we propose that, by analogy to the influenza virus M2 channel, membrane permeabilization and the resultant acidification of the arenavirus interior facilitate viral fusion and/or post-fusion steps of infection.

microbiology↗

bioRxiv2022

Phosphatidylserine within the Viral Membrane Enhances Chikungunya Virus Infectivity in a Cell-type Dependent Manner

Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host. ImportanceOutbreaks of Chikungunya virus (CHIKV) have occurred throughout Africa, Asia, and Europe. Climate change permits the expansion of Aedes mosquito vectors into more temperate regions, broadening the geographic range and increasing the frequency of future human outbreaks. The molecular basis underlying the broad host and cellular tropism of CHIKV remains unresolved. While several host molecules have been implicated in CHIKV viral attachment and entry, the role of lipid-mediated attachment (viral apoptotic mimicry) is unclear. We observed that higher levels of externalized phosphatidylserine (PS) in the viral lipid bilayer correlated with enhanced CHIKV infectivity in mammalian cells abundant with PS receptors and lacking alternative attachment factors. Interestingly, CHIKV infection in mosquito Aag2 cells was also affected by viral PS accessibility. This study further delineates the role of virus-cell attachment molecules in CHIKV infection. Viral apoptotic mimicry has potential to influence CHIKV dynamics in vivo in both the human and mosquito host.

microbiology↗

bioRxiv2021

Modulation of immunosuppressant drug treatment to improve SARS-CoV-2 vaccine efficacy in mice

The COVID-19 pandemic dramatically demonstrated the need for improved vaccination strategies and therapeutic responses to combat infectious diseases. However, the efficacy of vaccines has not yet been demonstrated in combination with commonly used immunosuppressive drug regimens. We sought to determine how common pharmaceutical drugs used in autoimmune disorders can alter immune responses to the SARS-CoV-2 spike protein vaccination. We treated mice with five immunosuppressant drugs (cyclophosphamide, leflunomide, methotrexate, methylprednisolone, and mycophenolate mofetil), each with various mechanisms of action prior to and following immunization with SARS-CoV-2 spike protein. We assessed the functionality of antibody responses to spike protein and compared immune cell populations in mice that received no treatment with those that received continuous or temporarily suspended immune suppressive therapy. All tested immunosuppressants significantly reduced the antibody titers in serum and functional antibody response against SARS-CoV-2 spike protein in immunized mice. Temporarily halting selected immunosuppressants (methylprednisolone and methotrexate, but not cyclophosphamide) improved antibody responses significantly. Through proof-of-principle experiments utilizing a mouse model, we demonstrated that immune suppression in autoimmune disorders through pharmaceutical treatments may impair vaccine response to SARS-CoV-2, and temporary suspension of immunosuppressant treatment may be necessary to mount an effective antibody vaccine response. This work provides feasibility for future clinical assessment of the impact of immunosuppressants on vaccine efficacy in humans. Significance StatementImmunosuppressant regimens are widely used as therapies for a variety of diseases, including autoimmune, inflammatory, and cancer. However, immunosuppressants can impair critical immune responses to vaccination. The impact of standard immunosuppressant use on the critical, developing SARS-CoV-2 vaccination strategies has not been well-described. In this study, we use a mouse model to determine how different immunosuppressant drugs that act through different mechanisms can impair the antibody response to SARS-CoV-2 spike protein, and how modulating these drug regimens may restore antibody levels and function.

immunology↗

bioRxiv2021

Cool temperatures limit Zika virus genome replication

Zika virus is a mosquito-borne flavivirus known to cause severe birth defects and neuroimmunological disorders. We have previously demonstrated that mosquito transmission of Zika virus decreases with temperature. While transmission was optimized at 29{degrees}C, it was limited at cool temperatures (< 22{degrees}C) due to poor virus establishment in the mosquitoes. Temperature is one of the strongest drivers of vector-borne disease transmission due to its profound effect on ectothermic mosquito vectors, viruses, and their interaction. Although there is substantial evidence of temperature effects on arbovirus replication and dissemination inside mosquitoes, little is known about whether temperature affects virus replication directly or indirectly through mosquito physiology. In order to determine the mechanisms behind temperature-induced changes in Zika virus transmission potential, we investigated different steps of the virus replication cycle in mosquito cells (C6/36) at optimal (28{degrees}C) and cool (20{degrees}C) temperatures. We found that cool temperature did not alter Zika virus entry or translation but reduced the amount of double-stranded RNA replication intermediates. If replication complexes were first formed at 28{degrees}C and the cells were subsequently shifted to 20{degrees}C, the late steps in the virus replication cycle were efficiently completed. These data suggest that cool temperature decreases the efficiency of Zika virus genome replication in mosquito cells. This phenotype was observed in the Asian-lineage of Zika virus, while the African-lineage Zika virus was less restrictive at 20{degrees}C. ImportanceWith half of the human population at risk, arboviral diseases represent a substantial global health burden. Zika virus, previously known to cause sporadic infections in humans, emerged in the Americas in 2015 and quickly spread worldwide. There was an urgent need to better understand the disease pathogenesis, develop therapeutics and vaccines, as well as to understand, predict, and control virus transmission. In order to efficiently predict the seasonality and geography for Zika virus transmission, we need a deeper understanding of the host-pathogen interactions and how they can be altered by environmental factors such as temperature. Identifying the step in the virus replication cycle that is inhibited in cool conditions can have implications in modeling the temperature suitability for arbovirus transmission as global environmental patterns change. Understanding the link between pathogen replication and environmental conditions can potentially be exploited to develop new vector control strategies in the future.

microbiology↗

bioRxiv2021

Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function

-Dystroglycan (-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xyl1,3-GlcA{beta}1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either -DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

biochemistry↗

bioRxiv2020

Ebola virus requires phosphatidylserine scrambling activity for efficient budding and optimal infectivity

AbstractEbola virus (EBOV) interacts with cells using two categories of cell surface receptors, C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic bodies. Many viruses coated with PS-containing lipid envelopes, acquired during budding from host cells, can also exploit these receptors for internalization. PS is restricted to the inner leaflet of the plasma membrane in homeostatic cells, an orientation that would be unfavorable for PS receptor-mediated uptake if conserved on the viral envelope. Therefore, it is theorized that viral infection induces host cell PS externalization to the outer leaflet during replication. Cells have several membrane scramblase enzymes that enrich outer leaflet PS when activated. Here, we investigate two scramblases, TMEM16F and XKR8, as possible mediators of cellular and viral envelope surface PS levels during recombinant VSV/EBOV-GP replication and EBOV virus-like particle (VLP) production. We found that rVSV/EBOV-GP and EBOV VLPs produced in XKR8 knockout cells contain decreased levels of PS in their outer leaflets. {Delta}XKR8-made rVSV/EBOV-GP is 70% less efficient at infecting cells through apoptotic mimicry compared to viruses made in parental cells. Our data suggest that virion surface PS acquisition requires XKR8 activity, whereas TMEM16F activity is not essential. Unexpectedly, we observed defective rVSV/G, rVSV/EBOV-GP, and EBOV VLP budding in {Delta}XKR8 cells, suggesting that phospholipid scrambling via XKR8 enhances both Ebola infectivity and budding efficiency. Overexpression of XKR8 dramatically increased budding activity, suggesting outer leaflet PS is required for both particle production and increased infectivity. ImportanceThe Democratic Republic of the Congo experienced its deadliest Ebola outbreak from 2018 to 2020, with 3,444 confirmed cases and 2,264 deaths (as of March 12, 2020). Owing to the extensive damage that these outbreaks have caused in Africa, as well as its future epidemic potential, Ebola virus (EBOV) ranks among the top eight priority pathogens outlined by the WHO in 2018. A comprehensive understanding of Ebola entry pathways into target cells is critical for antiviral development and outbreak control. Thus far, host-cell scramblases TMEM16F and XKR8 have each been named as the sole mediator of Ebola envelope surface phosphatidylserine (PS). We assessed the contributions of these proteins using CRISPR knockout cells and two EBOV models: rVSV/EBOV-GP and EBOV VLPs. We observed that XKR8 is required for optimal EBOV envelope PS levels, PS receptor engagement, and particle budding across all viral models, whereas TMEM16F did not play a major role.

microbiology↗

bioRxiv2019

Effects of temperature on Zika dynamics and control

ABSTRACTWhen a formerly rare pathogen emerges to cause a pandemic, it is critical to understand the ecology of the disease dynamics and its potential effects on disease control. Here, we take advantage of newly available experimental data to parameterize a temperature-dependent dynamical model of Zika virus (ZIKV) transmission, and analyze the effects of temperature variability and the parameters related to control strategies on ZIKV R0 and the final epidemic size (i.e., total number of human cases). Sensitivity analyses identified that R0 and the final epidemic size were largely driven by different parameters, with the exception of temperature, which is the dominant driver of epidemic dynamics in the models. Our estimate of R0 had a single optimum temperature (≈ 30° C), comparable to recently published results (≈ 29°)1. However, the total number of human cases (“final epidemic size”) is maximized across a wider temperature range, from 24 to 36°C. The models indicate that the disease is highly sensitive to seasonal temperature variation. For example, although the model predicts that Zika transmission cannot occur at a constant temperature of 22°C, with seasonal variation of 5°C around a mean of 22°C, the model predicts a larger epidemic than what would occur at a constant 30°C, the temperature predicted to maximize R0. This suggests that the potential geographic range of Zika is wider than indicated from static R0 models, underscoring the importance of climate dynamics and variation on emerging infectious diseases.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2018

Impacts of temperature on Zika virus transmission potential: combining empirical and mechanistic modeling approaches

Temperature is a strong driver of vector-borne disease transmission. Yet, for emerging arboviruses we lack fundamental knowledge on the relationship between transmission and temperature. Current models rely on the untested assumption that Zika virus responds similarly to dengue virus, potentially limiting our ability to accurately predict the spread of Zika. We conducted experiments to estimate the thermal performance of Zika virus (ZIKV) in field-derived Aedes aegypti across eight constant temperatures. We observed strong, unimodal effects of temperature on vector competence, extrinsic incubation period, and mosquito survival. We used thermal responses of these traits to update an existing temperature-dependent model to infer temperature effects on ZIKV transmission. ZIKV transmission was optimized at 29{degrees}C, and had a thermal range of 22.7{degrees}C - 34.7{degrees}C. Thus, as temperatures move toward the predicted thermal optimum (29{degrees}C) due to climate change, urbanization, or seasonally, Zika could expand north and into longer seasons. In contrast, areas that are near the thermal optimum were predicted to experience a decrease in overall environmental suitability. We also demonstrate that the predicted thermal minimum for Zika transmission is 5{degrees}C warmer than that of dengue, and current global estimates on the environmental suitability for Zika are greatly over-predicting its possible range.

ecology↗

bioRxiv2017

Estimating the effects of variation in viremia on mosquito susceptibility, infectiousness, and R0 of Zika in Aedes aegypti

Zika virus (ZIKV) is an arbovirus primarily transmitted by Aedes mosquitoes. Like most viral infections, ZIKV viremia varies over several orders of magnitude, with unknown consequences for transmission. To determine the effect of viral concentration on ZIKV transmission risk, we exposed field-derived Ae. aegypti mosquitoes to four doses (103, 104, 105, 106 PFU/mL) representative of potential variation in the field. We demonstrate that increasing ZIKV dose in the blood-meal significantly increases the probability of mosquitoes becoming infected and infectious, as well as the rate at which virus spreads to the saliva, but found no effect on dissemination efficiency or mosquito mortality. We also demonstrate that determining infection using RT-qPCR approaches rather than plaque assays potentially over-estimates key pathogen parameters, including the time at which mosquitoes become infectious and viral burden. Finally, using these data to parameterize an R0 model, we demonstrate that variation in viremia substantially affects transmission risk.

microbiology↗

bioRxiv2017

Carry-over effects of larval microclimate on the transmission potential of a mosquito-borne pathogen

Climate shapes the transmission of mosquito-borne pathogens through impacts on both the vector and the pathogen. In addition to direct effects of the environment, carry-over effects from previous life history stages can influence mosquito traits relevant to disease transmission. While this has been explored in a laboratory setting, the net effect of temperature-mediated carry-over effects due to relevant environmental variation in the larval stage is ambiguous. Here, we use data collected from a semi-field experiment investigating dengue dynamics in Aedes albopictus across a natural environmental gradient to parameterize a dengue transmission model. We reared Ae. albopictus across three different land classes characterized by their proportion of impervious surface. Emerged females were offered a dengue infectious bloodmeal, kept at a constant 27 {degrees}C, and assayed for infection, dissemination, and infectiousness 21 days post infection. Incorporating carry-over effects of larval environment on measures of vector competence resulted in lower predicted dengue transmission potential across land class and season, however a strong positive relationship with larval environmental temperature remained. Given the significant impact of carry-over effects, future mechanistic models of disease transmission should include both direct and carry-over effects of environmental temperature.

ecology↗