Cellular Entry Pathways Research Articles

Endocytosis Pathways of Graphene Quantum Dots

Graphene quantum dots (GQDs) have emerged as a forerunner of carbon nanbiootechnology due to their multifunctional delivery and imaging capabilities as they exhibit fluorescence in the visible and near-infrared, high biocompatibility, and water solubility. These properties put GQDs forward as a compelling drug delivery platform that has already been utilized in a variety of applications including the delivery of chemotherapeutics, antibiotics as well as siRNA and CRISPR-based gene therapy. However, cellular entry pathways of this nanomaterial still remain largely undefined. In a number of studies describing GQD cellular internalization different and, often, conflicting results have been presented due to surveying only few endocytosis inhibitors and disregarding their potential off-target pathways. Understanding the cell internalization routes of GQDs is crucial while delivering drugs in different types of cell lines. Herein, we performed a holistic approach to cell uptake studies on GQDs of different charges by the comparative study of their preferred endocytosis paths in non-cancerous (HEK-293) and cancerous (HeLa) cell lines. The concentration and cell viability of GQDs were determined by MTT assays, while their endocytosis paths were investigated through confocal fluorescence microscopy on cells treated for up to 24 hours. The potential for GQD interactions with the cell membrane was also examined via zeta (ζ) potential measurements. Our findings provide insights into the internalization mechanisms of the GQDs into cell membranes of healthy and cancer cells. The optimization of these mechanisms can serve for the enhancement of a variety of novel GQD applications in biomedicine including therapeutic delivery, disease detection through sensing as well as diagnostic imaging.

Micropterus salmoides rhabdovirus enters cells via clathrin-mediated endocytosis pathway in a pH-, dynamin-, microtubule-, rab5-, and rab7-dependent manner.

Although Micropterus salmoides rhabdovirus (MSRV) causes serious fish epidemics worldwide, the detailed mechanism of MSRV entry into host cells remains unknown. Here, we comprehensively investigated the mechanism of MSRV entry into epithelioma papulosum cyprinid (EPC) cells. This study demonstrated that MSRV enters EPC cells via a low pH, dynamin-dependent, microtubule-dependent, and clathrin-mediated endocytosis. Subsequently, MSRV transports from early endosomes to late endosomes and further into lysosomes in a microtubule-dependent manner. The characterization of MSRV entry will further advance the understanding of rhabdovirus cellular entry pathways and provide novel targets for antiviral drug against MSRV infection.

Enhanced sucrose-mediated cryoprotection of siRNA-loaded poly (lactic-co-glycolic acid) nanoparticles

The present study aimed to determine the effects of sucrose on the physical stability, cellular entry pathways and functional efficacy of poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs). PLGA-NPs were synthesized in the absence or presence of 10 % sucrose, using HEI-101, an unmodified small interfering RNA (siRNA), as a drug model. The newly synthesized HEI-101-loaded PLGA-NPs (HEI-101-NPs) were exposed to repeated freeze-thaw cycles and iteratively tested over a six-month evaluation period. The effect of sucrose stabilization on HEI-101-NPs was independently tested in vitro for biocompatibility and cellular uptake in IMO-2B1 cells. Data analyses suggest that, without sucrose, freeze-thaw cycles of HEI-101-NPs resulted in increased particle diameter, increased polydispersity index, and reduced zeta potential. In contrast, a substantial improvement in the physical stability of HEI-101-NPs was observed in the presence of 10 % sucrose. The data revealed that the release of HEI-101 from the PLGA-NPs was governed by polymer erosion and drug diffusion. Data from cellular uptake study in IMO-2B1 cells demonstrated that, 10 % sucrose significantly reduced the inhibitory effect of nocodazole on the microtubule-dependent uptake of PLGA-NPs. In addition, the presence of 10 % sucrose seemed to lessen the inhibitory effect of sodium azide on the energy-dependent uptake of PLGA-NPs. Overall, the current data suggest that the cellular internalization of PLGA-NPs occurred through the polymerization of actin filaments under the control of the microtubules. Our findings reveal cryoprotective effect of 10 % sucrose on HEI-101-NPs that confers marked improvements in the stability, cellular uptake and efficiency for the delivery of biomolecules to inner ear cells.

Interplay of Ferritin Accumulation and Ferroportin Loss in Ageing Brain: Implication for Protein Aggregation in Down Syndrome Dementia, Alzheimer's, and Parkinson's Diseases.

Iron accumulates in the ageing brain and in brains with neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Down syndrome (DS) dementia. However, the mechanisms of iron deposition and regional selectivity in the brain are ill-understood. The identification of several proteins that are involved in iron homeostasis, transport, and regulation suggests avenues to explore their function in neurodegenerative diseases. To uncover the molecular mechanisms underlying this association, we investigated the distribution and expression of these key iron proteins in brain tissues of patients with AD, DS, PD, and compared them with age-matched controls. Ferritin is an iron storage protein that is deposited in senile plaques in the AD and DS brain, as well as in neuromelanin-containing neurons in the Lewy bodies in PD brain. The transporter of ferrous iron, Divalent metal protein 1 (DMT1), was observed solely in the capillary endothelium and in astrocytes close to the ventricles with unchanged expression in PD. The principal iron transporter, ferroportin, is strikingly reduced in the AD brain compared to age-matched controls. Extensive blood vessel damage in the basal ganglia and deposition of punctate ferritin heavy chain (FTH) and hepcidin were found in the caudate and putamen within striosomes/matrix in both PD and DS brains. We suggest that downregulation of ferroportin could be a key reason for iron mismanagement through disruption of cellular entry and exit pathways of the endothelium. Membrane damage and subsequent impairment of ferroportin and hepcidin causes oxidative stress that contributes to neurodegeneration seen in DS, AD, and in PD subjects. We further propose that a lack of ferritin contributes to neurodegeneration as a consequence of failure to export toxic metals from the cortex in AD/DS and from the substantia nigra and caudate/putamen in PD brain.

Antibody Response to the Coronavirus Disease 2019 Ad26.COV2.S Vaccine Among Maintenance Dialysis Patients

Antibody Response to the Coronavirus Disease 2019 Ad26.COV2.S Vaccine Among Maintenance Dialysis Patients

β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that β-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of β-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. β-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg2+ and divalent metal cations such as Ca2+, Zn2+, Cd2+, Mn2+, and Ni2+. TRPM7 channels are inhibited by cytosolic Mg2+, rendering them largely inactive in intact cells. The dependence of channel activity on extracellular Mg2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg2+ concentrations varying between 400 nM and 1.4 mM for 1-3 days, obtaining an IC50 value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg2+]o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg2+ depletion. A total of 1.4 mM [Mg2+]o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg2+ depletion but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni2+, Zn2+, Cd2+, Co2+, Mn2+, Sr2+, and Ba2+ requires TRPM7 channel activity. Mg2+ loading modestly reduced cytotoxicity of Zn2+, Co2+, Ni2+, and Mn2+ but not of Cd2+. Channel blocker NS8593 reduced Co2+ and Mn2+ but not Cd2+ or Zn2+ cytotoxicity and interfered with Mg2+ loading as evaluated by TRPM7 channel basal activity. Ba2+ and Sr2+ were neither detectably toxic nor permeant through the plasma membrane. These results indicate that in Jurkat T cells, entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg2+ entry requires TRPM7 channels.

β-Coronaviruses Use Lysosomal Organelles for Cellular Egress

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that β-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.Funding: NAB, SG, TDR, EP, QQ, MF and CB were supported with NHLBI/NIH; GAB and SRA were supported with NCI/NIH intramural funds. PMT was supported by NIH R01 A1091985-05; SP by NIH R01 NS36592 and AF by F32-AI113973; VH by NIH R37GM058615; GW by NIH R01AI35270.Conflict of Interest: None.

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens1. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and released from hepatocytes without lysis in small vesicles resembling exosomes2,3. These quasi-enveloped virions (eHAV) are infectious and the only form of virus detected in blood during acute infection2. By contrast, non-enveloped, naked virions (nHAV) are shed in feces, stripped of membranes by bile salts during passage through bile ducts to the gut4. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is enigmatic. Here we describe a genome-wide forward screen that identified glucosylceramide synthase (UGCG) and other components of the ganglioside synthetic pathway as crucial host factors required for cellular entry by hepatoviruses. We show that gangliosides, preferentially disialogangliosides, function as essential endolysosome receptors required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1+ endolysosomes. Gangliosides relieve this block, binding the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.

Length-Dependent Uptake, Inflammation, and Intracellular Processing of Single-Wall Carbon Nanotubes in Macrophages

Understanding the impact of single-wall carbon nanotube (SWCNT) lengths on cytotoxicity, uptake and intracellular processing is essential in realizing SWCNT-based drug delivery systems. In addition, cell type-specific response to SWCNT cannot be neglected since SWCNT uptake and processing vary across different cell types. Immune cell-specific processing of SWCNTs in different lengths is of particular interest because immune cells are capable of two distinct size-dependent cellular entry pathways; endocytosis and phagocytosis, and they engage in various physiological conditions such as wound healing, inflammation, and cancer. In this work, we present the impact of SWCNT lengths on uptake, inflammation, and intracellular processing of SWCNTs in macrophages. SWCNTs with average lengths of 50 nm (ultra-short), 145 nm (short), and 500 nm (long) were noncovalently dispersed with bovine serum albumin (BSA), which promotes cellular uptake without affecting cell viability. Interestingly, the amount of uptake was significantly higher for ultra-short SWCNTs compared to longer nanotubes. Moreover, short-SWCNTs became highly bundled in macrophages, which were mostly retained for at least 24 h. In contrast, most long- and ultra-short- SWCNTs remained individualized inside macrophages and were eventually exocytosed over time. Further, we observed pro-inflammatory behavior of macrophage upon exposure to ultra-short-SWCNTs. Overall, the length-dependent inflammation and intracellular processing in macrophages allow for tailoring SWCNT properties for biomedical therapy and imaging. High internalization and subsequent exocytosis of ultra-short-SWCNTs and the triggered pro-inflammatory behavior are more preferred for cancer therapy and drug delivery, while high retention of short-SWCNTs in macrophages could be more desirable for biomedical imaging and macrophage tracking in vivo.

Understanding the synergistic effect of physicochemical properties of nanoparticles and their cellular entry pathways

Gaining precise control over the cellular entry pathway of nanomaterials is key in achieving cytosolic delivery, accessing subcellular environments, and regulating toxicity. However, this precise control requires a fundamental understanding of the behavior of nanomaterials at the bio-nano interface. Herein, we report a computational study investigating the synergistic effect of several key physicochemical properties of nanomaterials on their cellular entry pathways. By examining interactions between monolayer-protected nanoparticles and model cell membranes in a three-dimensional parameter space of size, surface charge/pKa, and ligand chemistry, we observed four different types of nanoparticle translocation for cellular entry which are: outer wrapping, free translocation, inner attach, and embedment. Nanoparticle size, surface charge/pKa, and ligand chemistry each play a unique role in determining the outcome of translocation. Specifically, membrane local curvature induced by nanoparticles upon contact is critical for initiating the translocation process. A generalized paradigm is proposed to describe the fundamental mechanisms underlying the bio-nano interface.

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.

Towards Understanding KSHV Fusion and Entry.

How viruses enter cells is of critical importance to pathogenesis in the host and for treatment strategies. Over the last several years, the herpesvirus field has made numerous and thoroughly fascinating discoveries about the entry of alpha-, beta-, and gamma-herpesviruses, giving rise to knowledge of entry at the amino acid level and the realization that, in some cases, researchers had overlooked whole sets of molecules essential for entry into critical cell types. Herpesviruses come equipped with multiple envelope glycoproteins which have several roles in many aspects of infection. For herpesvirus entry, it is usual that a collective of glycoproteins is involved in attachment to the cell surface, specific interactions then take place between viral glycoproteins and host cell receptors, and then molecular interactions and triggers occur, ultimately leading to viral envelope fusion with the host cell membrane. The fact that there are multiple cell and virus molecules involved with the build-up to fusion enhances the diversity and specificity of target cell types, the cellular entry pathways the virus commandeers, and the final triggers of fusion. This review will examine discoveries relating to how Kaposi’s sarcoma-associated herpesvirus (KSHV) encounters and binds to critical cell types, how cells internalize the virus, and how the fusion may occur between the viral membrane and the host cell membrane. Particular focus is given to viral glycoproteins and what is known about their mechanisms of action.

Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity.

Alpha interferon (IFN-α) induces the transfer of resistance to hepatitis B virus (HBV) from liver nonparenchymal cells (LNPCs) to hepatocytes via exosomes. However, little is known about the entry machinery and pathway involved in the transmission of IFN-α-induced antiviral activity. In this study, we found that macrophage exosomes uniquely depend on T cell immunoglobulin and mucin receptor 1 (TIM-1), a hepatitis A virus (HAV) receptor, to enter hepatocytes for delivering IFN-α-induced anti-HBV activity. Moreover, two primary endocytic routes for virus infection, clathrin-mediated endocytosis (CME) and macropinocytosis, collaborate to permit exosome entry and anti-HBV activity transfer. Subsequently, lysobisphosphatidic acid (LBPA), an anionic lipid closely related to endosome penetration of virus, facilitates membrane fusion of exosomes in late endosomes/multivesicular bodies (LEs/MVBs) and the accompanying exosomal cargo uncoating. Together, our findings provide comprehensive insights into the transmission route of macrophage exosomes to efficiently deliver IFN-α-induced antiviral substances and highlight the similarities between the entry mechanisms of exosomes and virus.IMPORTANCE Our previous study showed that LNPC-derived exosomes could transmit IFN-α-induced antiviral activity to HBV replicating hepatocytes, but the concrete transmission mechanisms, which include exosome entry and exosomal cargo release, remain unclear. In this study, we found that virus entry machinery and pathway were also applied to exosome-mediated cell-to-cell antiviral activity transfer. Macrophage-derived exosomes distinctively exploit hepatitis A virus receptor for access to hepatocytes. Later, CME and macropinocytosis are utilized by exosomes, followed by exosome-endosome fusion for efficient transfer of IFN-α-induced anti-HBV activity. We believe that understanding the cellular entry pathway of exosomes will be beneficial to designing exosomes as efficient vehicles for antiviral therapy.

Systemic Delivery of Sirna-Based Therapeutics Using Single-Walled Carbon Nanotubes

Background: Carbon nanotubes (CNTs) are potential candidates for drug, antigen and nucleic acid delivery vehicle in nanomedicine. The large surface of CNTs provides structural advantages and allows loading of functional groups or therapeutics such as nucleic acid, drugs and proteins. Aim: Our study is to deliver siRNA to cells using single walled carbon nanotube (SWNT) to achieve gene silencing effect. Methods: SWNT was functionalized by dissolving 1 mg of HiPCo SWNTs and 5 mg of PL-PEG-NH or PL-PEG-maleimide in 5 mL of water, sonicated for 60 min at room temperature and centrifuged for 6 h. The supernatant were collected and measured their concentration at 808 nm by ultraviolet-VIS-NIR spectrometer. The resulted noncovalent functionalized SWNTs were further conjugated with a 5′-thiolated siRNA against GFP (siGFP) and RFP (siRFP). In vitro silencing of GFP and RFP expression by SWNT-siRNA were evaluated in stable expression cell lines by fluorescence spectroscopy. Results: A range of 50%–80% GFP expression knocked down was observed in H1299, HeLa, MCF-7 and 293T cells by SWNT-siGFP. SWNT conjugated with both siGFP and siRFP were shown knocking down both GFP and RFP simultaneously in H1299 stable coexpression cell line. Also, gene silencing was observed despite incubation with inhibitors on different cellular internalization pathways. They are chlorpromazine for clathrin-mediated endocytosis inhibitor, genistein for caveolae-mediated endocytosis inhibitor and sodium azide for energy depletion agent. We also found that the internalization of SWNT by the nonphagocytic cells (H1299) did not solely depend on single cellular entry pathway to achieve the gene silencing effect. Conclusion: The successful knockdown of GFP expression in different cell lines indicated that siRNA were released from the conjugated SWNT-siRNA in the cytoplasm and silent the gene expression. It is also indicated that 2 different types of siRNA targets could be conjugated with SWNT and achieved 2 different gene silencing effects simultaneously.

Cellular uptake of hepatitis B virus envelope L particles is independent of sodium taurocholate cotransporting polypeptide, but dependent on heparan sulfate proteoglycan

Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.

Revealing the Transport Cycle Dynamics of the Sodium Dependent Sugar Transporter by Double Electron-Electron Resonance and Wide-Angle X-ray Scattering

Dietary sugars have an immense importance in the metabolism of all cells. As such, it is vital to understand their cellular entry and exit pathways through specific transporters. Our research focuses on the sodium dependent sugar transporter from Vibrio parahemolyticus (vSGLT). Currently, this is the best structural model system for the mammalian transporters, which are responsible for sugar absorption in the intestine and reabsorption in the kidney. Recently, this transporter has been the target of various drugs that lower blood-sugar levels by inhibiting sugar reabsorption in the kidney.SGLTs are highly dynamic and switch between a number of conformations to facilitate sugar passage across the cell membrane. There are currently two available x-ray structures for vSGLT in an inward-occluded1 and an inward-open2 conformation. These two distinct conformations have fundamentally advanced our understanding of sugar transport to an atomic level, but did not encapsulate the dynamics of transport, nor the nature of the outward-open conformation.In order to gain further insight into the ligand dependent dynamics of vSGLT, we have conducted double electron-electron resonance (DEER) and wide-angle x-ray scattering (WAXS) studies under three conditions, each favoring different conformers in the protein: in the absence of sodium and galactose, in the presence of sodium only and in the presence of both sodium and galactose.We will discuss the overall insights gained by these two approaches, the similarities and differences with other transporters that share the Leu-T fold and the structural refinement that included combined energy terms from DEER and WAXS.1. Faham et al., (2008) Science 321(5890).2. Watanabe et al., (2010) Nature 468.

Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane.

ABSTRACTIntracellular signaling connected to integrin activation is known to induce cytoplasmic Ca2+ release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca2+ levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca2+ release, while EHV-4 with gH1 triggered significant Ca2+ release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca2+ release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca2+ release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane.

The endoplasmic reticulum membrane J protein C18 executes a distinct role in promoting simian virus 40 membrane penetration.

The nonenveloped simian virus 40 (SV40) hijacks the three endoplasmic reticulum (ER) membrane-bound J proteins B12, B14, and C18 to escape from the ER into the cytosol en route to successful infection. How C18 controls SV40 ER-to-cytosol membrane penetration is the least understood of these processes. We previously found that SV40 triggers B12 and B14 to reorganize into discrete puncta in the ER membrane called foci, structures postulated to represent the cytosol entry site (C. P. Walczak, M. S. Ravindran, T. Inoue, and B. Tsai, PLoS Pathog 10: e1004007, 2014). We now find that SV40 also recruits C18 to the virus-induced B12/B14 foci. Importantly, the C18 foci harbor membrane penetration-competent SV40, further implicating this structure as the membrane penetration site. Consistent with this, a mutant SV40 that cannot penetrate the ER membrane and promote infection fails to induce C18 foci. C18 also regulates the recruitment of B12/B14 into the foci. In contrast to B14, C18's cytosolic Hsc70-binding J domain, but not the lumenal domain, is essential for its targeting to the foci; this J domain likewise is necessary to support SV40 infection. Knockdown-rescue experiments reveal that C18 executes a role that is not redundant with those of B12/B14 during SV40 infection. Collectively, our data illuminate C18's contribution to SV40 ER membrane penetration, strengthening the idea that SV40-triggered foci are critical for cytosol entry. Polyomaviruses (PyVs) cause devastating human diseases, particularly in immunocompromised patients. As this virus family continues to be a significant human pathogen, clarifying the molecular basis of their cellular entry pathway remains a high priority. To infect cells, PyV traffics from the cell surface to the ER, where it penetrates the ER membrane to reach the cytosol. In the cytosol, the virus moves to the nucleus to cause infection. ER-to-cytosol membrane penetration is a critical yet mysterious infection step. In this study, we clarify the role of an ER membrane protein called C18 in mobilizing the simian PyV SV40, a PyV archetype, from the ER into the cytosol. Our findings also support the hypothesis that SV40 induces the formation of punctate structures in the ER membrane, called foci, that serve as the portal for cytosol entry of the virus.

Probing the Cellular Entry Pathway via TolC of the Cytotoxin, Colicin E1

The pathway of cellular entry of colicins and viral DNA is a fundamental structure problem that is relevant to an understanding the molecular basis of infectious diseases. The cytotoxin colicin E1 uses the outer membrane/trans-periplasmic drug export protein, TolC, for its import. TolC consists of a 12 strand OM β-barrel connected to a 12 strand α-helical tunnel that defines a pathway through the peptidoglycan barrier in the periplasm to the cytoplasmic membrane in which the C-terminal domain of colicin E1 inserts and forms a depolarizing ion channel. The nature of the interaction of the colicin with TolC and the mechanism of its translocation are unknown. Previous studies with planar bilayers showed that colicin E1 occludes TolC channels [1], as do certain colicin T-domain peptides [2].Here, in vivo protection of sensitive E. coli from colicin E1 by a series of N-terminal colicin peptides is used to probe the interaction of the colicin with TolC, with the goal of defining the sites of TolC-colicin interaction and the mechanism of colicin entry. N-terminal segments ‘1-40’,’1-81’, and ‘1-100’ of the colicin did not provide cytotoxic protection, nor occlude TolC channels. Segments ‘1-120’, ‘1-140’, 1-190’, as well as ‘41-190’ and ‘57-190’ protected efficiently in vivo and occluded TolC with high efficiency. Occlusion required a trans-negative electrical potential and was irreversible. Co-elution of the colicin peptides with TolC on a Superdex 200 column was shown for ‘41-190’, but not for ‘1-81.’ In addition to the correlation with protection in vivo from killing by colicin E1, occlusion efficiency also correlated with a basic pI between colicin residues 82 and 140. [1] Biophys. J., 87:3901-3911, 2004; [2] Biochem. Soc. Trans., 40:1463-1468, 2012. Support: NIH AI091633 (KSJ); NIH GM38323 and the Henry Koffler Professorship (WAC).