Kinetics Of Entry Research Articles

Natural Variation in HIV-1 Entry Kinetics Map to Specific Residues and Reveal an Interdependence Between Attachment and Fusion.

The first step of HIV-1 infection is entry, where virus-cell membrane fusion is driven by the HIV-1 envelope glycoprotein through a series of conformational changes. Some of the most broadly active entry inhibitors work by binding conformations that exist only transiently during entry. The lifetimes of these states and the kinetics of entry are important elements of inhibitor activity for which little is known. We demonstrate a remarkable range of kinetics among 257 diverse HIV-1 isolates and find that this phenotype is highly flexible, with multiple single-residue determinants. Examination of the kinetics of two conformational landmarks shed light on novel kinetic features that offer new details about the role of co-receptor engagement and provide a framework to explain entry inhibitor synergy.

Revealing membrane factors that control SARS-CoV-2 membrane fusion and entry kinetics

Revealing membrane factors that control SARS-CoV-2 membrane fusion and entry kinetics

Dynamics of macrophage tumor infiltration

Long-term remission in cancer patients treated with ex vivo bona fide M1-induced macrophages has been poor, and the reasons behind this are not understood. Injected M1 macrophages must physically migrate to tumors to execute their role that leads to a therapeutic benefit. However, the trafficking of macrophages to tumors has not been rigorously studied. We hypothesized that trafficking capabilities of macrophages are impacted when naïve M0 macrophages are converted into an M1 phenotype for macrophage therapy. To test this, we developed a three-dimensional assay comprising a tumor spheroid and macrophages to quantify macrophage tumor transport. Cell migration, permeability, and kinetics of tumor entry were quantitatively defined and compared between macrophage phenotypes. Our results demonstrate that compared to M0 macrophages, M1 macrophages migrate less efficiently toward the tumor spheroid and exhibit a fivefold lower tumor permeability. Live imaging data combined with unsupervised machine learning algorithms reveal that macrophage migration correlates with their shape transitions. Our studies highlight the importance of transport considerations in determining the efficacy of cell therapies. This study quantitatively demonstrates that the transport properties of macrophages in tumors depend on their phenotype.

Kinetic Analysis on Hydrogen Entry into Steel in Aqueous Solutions by Alternating Current Method

Degradation of steels due to the hydrogen embrittlement, which is related to the hydrogen penetration reaction (HPR) into steel, is a serious problem from the standpoint of infrastructure preservation. A Devanathan-Stachurski permeation technique [1, 2] is a well-known method to determine a hydrogen penetration rate in metals. In this technique, two independent electrochemical cells are arranged on right across two surfaces of thin metal sheet and anodic and cathodic polarizations can be performed with each cell using the three-electrode system. The hydrogen penetration rate can be evaluated by measuring the oxidation current of hydrogen atoms diffused in the metal sheet at the anode side. The electrochemical impedance spectroscopy (EIS) was used for electrochemical measurement with the Devanathan cell to investigate HPR mechanisms because the time constants of hydrogen evolution and entry can be discriminated [3-5]. In this study, we used a complex hydrogen penetration ratio (CHPR), which is a transfer function related to hydrogen entry and diffusion in metal sheet, with the Devanathan cell to investigate kinetics of hydrogen entry into metals. The CHPRs of carbon steel sheet immersed in aqueous solution of different pH’s were measured in this study. The rate constant of hydrogen entry was determined by comparing experimental results with the simulated ones according to the theoretical equation, and influence of pH on hydrogen entry was discussed.

Cell-Cell Fusion Assays to Study Henipavirus Entry and Evaluate Therapeutics.

Henipaviruses include the deadly zoonotic Nipah (NiV) and Hendra (HeV) paramyxoviruses, which have caused recurring outbreaks in human populations. A hallmark of henipavirus infection is the induction of cell-cell fusion (syncytia), caused by the expression of the attachment (G) and fusion (F) glycoproteins on the surface of infected cells. The interactions of G and F with each other and with receptors on cellular plasma membranes drive both viral entry and syncytia formation and are thus of great interest. While F shares structural and functional homologies with class I fusion proteins of other viruses such as influenza and human immunodeficiency viruses, the intricate interactions between the G and F glycoproteins allow for unique approaches to studying the class I membrane fusion process. This allows us to study cell-cell fusion and viral entry kinetics for BSL-4 pathogens such as NiV and HeV under BSL-2 conditions using recombinant DNA techniques. Here, we present approaches to studying henipavirus-induced membrane fusion for currently identified and emerging henipaviruses, including more traditional syncytia counting-based cell-cell fusion assay and a new heterologous fluorescent dye exchange cell-cell fusion assay.

Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.

The Golgi apparatus is a pivotal secretory organelle in membrane trafficking, a hub responsible for posttranslational modifications, sorting, and trafficking of newly synthetized proteins received from the endoplasmic reticulum (ER). Different protein cargoes have been shown to travel through the Golgi stacks with different kinetics. Dysregulated transport and altered residency time of cargoes in the Golgi can impair their functionality. To study the anterograde trafficking of specific protein cargoes, innovative molecular methods have been developed to synchronize the traffic of selected cargoes from the ER in live cells. These methods of synchronization now provide the ability to quantify the Golgi entry and exit kinetics of defined cargo. In this chapter, we describe a quantitative, accurate, and semiautomated protocol to image and quantify the anterograde trafficking of individual cargo traversing the Golgi. This protocol, using free software, is compatible with different synchronization techniques, and can be used for a range of applications, such as comparing the Golgi kinetics of (1) different cargoes, (2) wild-type cargo vs mutated cargo, (3) the same cargo under different Golgi conditions, and (4) cargoes in drug screening platforms. The method can also be applied to study the localization and transit of a cargo through different organelles other than the Golgi apparatus.

GAPDH, Interferon γ, and Nitric Oxide: Inhibitors of Coronaviruses

As the COVID-19 pandemic finishes its second year, progress has been made against SARS-CoV-2 with vaccine candidates showing efficacy against this latest coronavirus strain. However, this pandemic presents a unique opportunity to investigate anti-viral therapies given the likely probability of another outbreak. One possible (and perhaps unlikely) therapeutic target could be GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Studies have show that downregulation of GAPDH leads to a decrease in interferon gamma (IFNγ production (which is an important cytokine response against coronaviruses and viruses in general). In this light, the previous coronavirus strain (SARS-CoV) has actually been shown to downregulate GAPDH. Although perhaps better known for its role in glycolysis, GAPDH also plays a role in gene expression of a varied set of genes by binding to their mRNA to affect stability and thereby translation Moreover, GAPDH is also upregulated by nitric oxide (NO), an inhibitor against both SARS-CoV and SARS-CoV-2. Additionally, GAPDH has also been shown to be a negative transcriptional regulator of AT1R (angiotensin II receptor 1), which has been shown to bind ACE2 for eventual endocytosis of the complex implicating GAPDH's potential role in the kinetics of coronavirus entry as well in downstream inflammatory signaling resulting from AT1R activation. Lastly, another important role for GAPDH is its requirement in the assembly of the GAIT complex that is responsible for termination of translation of IFNγ-responsive genes that would be critical for the resolution of any inflammatory response. These observations would imply that sufficient levels of GAPDH are needed for immune responses to function properly during a coronaviral infection. By examining different coronavirus studies, this review explores GAPDH's role as an inhibitor of coronaviruses (at the viral transcriptional level and also as a modulator of gene expression related to inflammation), and its signal transduction links to the IFNγ and NO pathways.

PET Imaging of brain muscarinic receptors with 18F-Fluorobenzyl-Dexetimide: A first in human study

M1 and M4 muscarinic receptor (mAChR) agonists are under development for the treatment of schizophrenia, Alzheimer's and Parkinson's disease. We performed first-in-human PET imaging of mAChR with 18F-Fluorobenzyl-Dexetimide (FDEX) in 10 healthy participants (29.4±4.3yrs). Four underwent dynamic brain scanning for 240 min, and then six underwent static brain scans at 120 and 160-min post injection of 250 MBq of FDEX. Gjedde-Patlak graphical analysis was applied to determine the influx constant (Ki). Regional tissue ratios (SUVR) were calculated using the cerebellar cortex as the reference region. No adverse events were observed. The tracer showed good brain entry (∼4.2% ID at 5 min) but irreversible distribution kinetics over four hours in regions of high mAChR. Binding was consistent with the distribution of mAChR receptors with striatum > cortex > hippocampus >> thalamus >>> cerebellum with low variance in regional binding between subjects. Ki was 0.42±0.04 in the putamen, 0.27±0.01 in frontal cortex, 0.25±0.02 in the hippocampus and 0.10±0.01 in the thalamus. SUVR at 120 and 240 min. were highly correlated with these Ki values with R2 of 0.91 and 0.99 respectively. FDEX yields high quality brain images with uptake in the known distribution of mAChR with remarkably little variance between normal subjects.

Variations in Env at amino acids 328 and 330 affect HIV-1 replicative fitness and entry inhibitor sensitivity

While the variations in the HIV-1 Env V3 loop have been the focus of much research to explore its functional effect, how specific mutations of certain amino acids in the V3 loop affect viral fitness remains unclear. In this study, we evaluated a series of natural polymorphisms at positions 328 and 330 with different combinations of adjacent glycosylation sites in the HIV-1 subtype B backbone to address their role in replicative fitness and sensitivity to entry inhibitors based on analysis of env sequence frequency at the population level. Pairwise growth competition experiment showed that wild-type virus with major consensus amino acids obviously had higher replicative fitness (P < 0.001). A change at position 328 to a less frequently occurring amino acid, K, together with a mutated N332 glycosylation site harbored lower fitness and became more sensitive to coreceptor antagonists (AMD3100), fusion inhibitors (T20) and sCD4. A change at position 330 to a less frequently occurring amino acid, Y, together with a mutated N332 glycosylation site resulted in higher fitness and less sensitivity to entry inhibitors (T20, AMD3100, and sCD4), and viruses containing both changes showed intermediate activity. It seems that the H330Y mutation compensated for the reduced replicative capacity caused by the Q328 K mutation. Moreover, viruses that showed lower replicative fitness also exhibited slower entry kinetics, lower levels of replication intermediates and protein packaging, and a lower ability to replicate in primary peripheral blood mononuclear cells (PBMCs). The findings highlight the functional effect of variations at 328 and 330 in the V3 loop on replicative fitness and may benefit HIV-1 treatment by helping predict the sensitivity to entry inhibitors.

Large-Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca*.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter‐pair‐based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate‐limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage‐dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1–2 events per second and long dwell times, in agreement with the liposome study. Applied‐field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 μm Ptm leads to a translocation rate of about one molecule per second and per channel.

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins.

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle.

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.

Coronaviruses: Is Sialic Acid a Gate to the Eye of Cytokine Storm? From the Entry to the Effects.

Coronaviruses (CoVs) are a diverse family of the enveloped human and animal viruses reported as causative agents for respiratory and intestinal infections. The high pathogenic potential of human CoVs, including SARS-CoV, MERS-CoV and SARS-CoV-2, is closely related to the invasion mechanisms underlying the attachment and entry of viral particles to the host cells. There is increasing evidence that sialylated compounds of cellular glycocalyx can serve as an important factor in the mechanism of CoVs infection. Additionally, the sialic acid-mediated cross-reactivity with the host immune lectins is known to exert the immune response of different intensity in selected pathological stages. Here, we focus on the last findings in the field of glycobiology in the context of the role of sialic acid in tissue tropism, viral entry kinetics and immune regulation in the CoVs infections.

High-Resolution Histological Landscape of AAV DNA Distribution in Cellular Compartments and Tissues following Local and Systemic Injection

Adeno-associated virus (AAV) is one of the most important gene delivery vehicles for in vivo gene therapy. Intramuscular (i.m.) and intravascular (i.v.) injection are commonly used for AAV gene transfer. Unfortunately, the fate of AAV vectors following administration remains unclear at the histological level. Taking advantage of RNAscope, a recently developed in situ hybridization technique that can reveal high-resolution viral DNA localization information, in this study, we evaluated body-wide distribution of an AAV9 vector in the context of the cell and tissue microenvironments. We observed distinctive kinetics of cell and nuclear entry of the AAV DNA in striated muscle and liver following i.m. and i.v. injection. We also found characteristic distribution patterns of the AAV DNA in various histological structures in internal organs, including gonads and lymph nodes, following i.v. injection. Finally, we showed significantly body-wide spreading of the AAV DNA following i.m. injection. These results add a new dimension to our understanding of AAV transduction biology and provide a basis for assessing the full impact of AAV gene therapy.

Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus.

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.

Chemical profiling of HIV-1 capsid-targeting antiviral PF74

The capsid protein (CA) of HIV-1 plays essential roles in multiple steps of the viral replication cycle by assembling into functional capsid core, controlling the kinetics of uncoating and nuclear entry, and interacting with various host factors. Targeting CA represents an attractive yet underexplored antiviral approach. Of all known CA-targeting small molecule chemotypes, the peptidomimetic PF74 is particularly interesting because it binds to the same pocket used by a few important host factors, resulting in highly desirable antiviral phenotypes. However, further development of PF74 entails understanding its pharmacophore and mitigating its poor metabolic stability. We report herein the design, synthesis, and evaluation of a large number of PF74 analogs aiming to provide a comprehensive chemical profiling of PF74 and advance the understanding on its detailed binding mechanism and pharmacophore. The analogs, containing structural variations mainly in the aniline domain and/or the indole domain, were assayed for their effect on stability of CA hexamers, antiviral activity, and cytotoxicity. Selected analogs were also tested for metabolic stability in liver microsomes, alone or in the presence of a CYP3A inhibitor. Collectively, our studies identified important pharmacophore elements and revealed additional binding features of PF74, which could aid in future design of improved ligands to better probe the molecular basis of CA-host factor interactions, design strategies to disrupt them, and ultimately identify viable CA-targeting antiviral leads.

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Nanocarrier anticancer drug-conjugates cause higher cellular deformations: culpable for mischief

Here we report nanocarrier-anticancer drug conjugates culpable for cellular deformations, critically evidenced through image-based analysis as a measure of karyoplasmic ratio (KR) and nuclear surface area (NSA). Multiwalled carbon nanotubes (MWCNTs) were coordinated additionally with Fe3O4 nanoparticles (NPs) to evaluate the symbiotic influence, and further conjugated to Dox for evaluating the cellular kinetics and for measuring cell deformations. Cellular entry kinetics of the CNT (CNT-Dox and CNT-Cys-Fe3O4-Dox) nanocarriers and their efficiency in nuclear localization were evaluated using cervical cancer (HeLa) cells. Of note, the Dox-bound nanocarriers showed significantly enhanced cell toxicity over the free form of the drug. CNT-Dox and CNT-Cys-Fe3O4-Dox influx occurred within 4 hours, while maximum cellular retention of Dox was observed for CNT-Dox at 24 h. However, the highest KR (∼0.51) was observed for CNT-Dox within 8 hours indicating similar cellular deformations using nanocarrier anticancer drug-conjugates to that of free Dox (KR ∼0.50) at 4 hours. In addition, we observed increased NSA at 4 h in Dox treatment whereas in the case of the Dox conjugated nanocarrier, increased NSA was noted at 8 h treatment. At 8 h exposure of HeLa cells with Dox conjugates, we observed that the cells fall into distinct regions of the morphospace with respect to KR and NSA. Conclusively, nano delivery systems considered for clinical and biomedical translations must take into account the possible negative influences imparting higher cellular deformations and secondary adverse effects over the free form of the drug.

Kinetic study of the hydrogen charging reaction on the graphite e in aqueous solution and in room temperature ionic liquid (RTIL)

Electrochemical impedance spectroscopy and other electrochemical methods were used to study the kinetics of hydrogen entry into porous, isotropic graphite and a single crystal graphite surfaces upon cathodic hydrogen charging and to calculate the hydrogen adsorption efficiencies. This model was first applied to graphite in an aqueous, alkaline solution, then in a room-temperature ionic liquid (RTIL), and, ultimately will be applied to FliNaK molten salt systems in future work. The results demonstrate that our model accurately describes the entry of hydrogen into graphite in harsh environments, which are important for the development of molten salt reactors (MSRs).

Human Abuse Potential of Oral NKTR-181 in Recreational Opioid Users: A Randomized, Double-Blind, Crossover Study.

To evaluate the human abuse potential, pharmacokinetics, pharmacodynamics, and safety of oral NKTR-181 (oxycodegol), a novel full mu-opioid receptor agonist, relative to oral oxycodone. This double-blind, randomized, single-dose, crossover human abuse potential study was conducted in healthy, adult, non-physically dependent recreational opioid users. Inpatient clinical research site. Seventy-one subjects randomized (95.7% male, 65.2% African American, mean age = 31.7 years). The primary objective was to compare two therapeutic doses of NKTR-181 (400 and 600 mg) with 40 and 60 mg of oxycodone and a supratherapeutic dose (1200 mg) of NKTR-181 with 60 mg of oxycodone using visual analog scale (VAS) ratings for Drug Liking "at this moment" (Drug Liking). Secondary objectives included VAS ratings for other subjective measures, and central nervous system (CNS) mu-opioid effects were assessed using pupillometry. Each subject received single oral doses of five treatments and matching placebo. Compared with 40 and 60 mg of oxycodone, the maximum mean Drug Liking score at 400 and 600 mg NKTR-181 was significantly lower, and the rate of onset and extent of Drug Liking for all NKTR-181 doses in the first two hours postdose were also significantly lower. Delayed attenuated Drug Liking and pupillary miosis response following administration of NKTR-181 vs oxycodone were consistent with slower NKTR-181 CNS entry kinetics and mu-opioid receptor binding. No adverse events were rated as severe, and somnolence and dizziness occurred more frequently when subjects received oxycodone. NKTR-181 at oral doses of 400 and 600 mg showed significantly fewer and less severe subjective effects accepted as representative of opioid abuse potential, such as lower peak Drug Liking in recreational opioid users, than 40 and 60 mg of oxycodone.