Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells.     

Imaging and Modeling the Dynamics of Clathrin-Mediated Endocytosis

Clathrin-mediated endocytosis (CME) plays a central role in cellular homeostasis and is mediated by clathrin-coated pits (CCPs). Live-cell imaging has revealed a remarkable heterogeneity in CCP assembly kinetics, which can be used as an intrinsic source of mechanistic information on CCP regulation but also poses several major problems for unbiased analysis of CME dynamics. The backbone of unveiling the molecular control of CME is an imaging-based inventory of the full diversity of individual CCP behaviors, which requires detection and tracking of structural fiduciaries and regulatory proteins with an accuracy of >99.9%, despite very low signals. This level of confidence can only be achieved by combining appropriate imaging modalities with self-diagnostic computational algorithms for image analysis and data mining.Clathrin-mediated endocytosis (CME) drives the uptake of diverse receptor-bound macromolecules and is one of the main endocytic mechanisms constitutively active in all mammalian cells. Clathrin-coated vesicles (CCVs) were the first transport vesicles to be isolated (Pearse 1975), which subsequently led to the identification of clathrin and the heterotetrameric adaptor protein AP2 as the major coat components (Pearse 1976, 1978). Further research in this area was spurred by the discovery that familial hypercholesterolemia is caused by a single substitution of a cysteine for a tyrosine in the cytoplasmic tail of the low-density lipoprotein receptor (LDLR), which disrupts its endocytic internalization motif and prevents its concentration in clathrin-coated pits (CCPs) (Anderson et al. 1977). In the following decades, biochemistry combined with molecular biology and electron microscopy (EM) have revealed much about the molecular players involved in CME (reviewed by Conner and Schmid 2003; Schmid and McMahon 2007; McMahon and Boucrot 2011; Boettner et al. 2012). Today, we know that CME is initiated via assembly of clathrin and AP2 to form CCPs and that receptor–ligand complexes (referred to as “cargo”) are concentrated in CCPs via direct interactions between endocytic motifs within their cytoplasmic domains and adaptor molecules that recruit clathrin. With the aid of a multitude of endocytic accessory proteins (EAPs)—many with as-yet poorly defined functions—CCPs undergo stabilization, maturation, and invagination. Finally, membrane fission, catalyzed by the GTPase dynamin, pinches off the CCV carrying its cargo into the cell.Although powerful and invaluable, bulk biochemical assays can only report cumulative and ensemble-averaged effects on CME, whereas EM only provides static snapshots of highly dynamic structures. Both approaches are not sufficient to resolve critical, rate-limiting stages of CCP maturation and alternative outcomes that prevent CCV internalization. They are also not sufficient to probe the frequently overlapping functions of individual components in CCP formation and maturation. Perturbation of molecular players in a system with such redundancy may lead to no detectable shifts, or to detectable shifts that merely represent system adaptation, and thus may not reveal the actual function of the targeted component itself. Moreover, perturbing CME may globally interfere with cell homeostasis, which can also elicit phenotypes unrelated to the actual functions of the target. To remedy these issues, it is necessary to follow the dynamics of CME at the level of individual CCPs and to correlate these behaviors with differential patterns of cargo and EAP recruitment and activity.These goals became approachable with the “GFP revolution” in the 1990s, which was paralleled by leaps in the sensitivity of digital light microscopy. For CME, the power of these technologies was first shown by Keen and colleagues, who used a green fluorescent protein (GFP) fusion of the clathrin light chain (CLC) to image clathrin dynamics by time-lapse wide-field epifluorescence microscopy (Gaidarov et al. 1999). Since then, numerous live-cell imaging studies have revealed remarkable heterogeneity in CCP assembly kinetics and internalization (Rappoport and Simon 2003; Ehrlich et al. 2004; Keyel et al. 2004; Merrifield et al. 2005; Loerke et al. 2009; Taylor et al. 2011). Although the physiological and molecular bases for this heterogeneity remain to be uncovered, the working hypothesis is that CCP heterogeneity arises from variations in molecular composition, in cortical membrane mechanics, and in differences between cell types. More recent advances in imaging and computational image data analyses have made it possible to determine the order in which EAPs are incorporated or released from growing CCPs. Thus, multidimensional live-cell imaging and mathematical models, in combination with very mild chemical, molecular, and mechanical perturbations, may uncover how the molecular composition of an assembling CCP affects its behavior. In the following we describe the developments of imaging modalities and image analysis methods that have led to the current state of the art in quantitative imaging of CME.     

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

Up to 60 different proteins are recruited to the site of clathrin-mediated endocytosis in an ordered sequence. These accessory proteins have roles during all the different stages of clathrin-mediated endocytosis. First, they participate in the initiation of the endocytic event, thereby determining when and where endocytic vesicles are made; later they are involved in the maturation of the clathrin coat, recruitment of specific cargo molecules, bending of the membrane, and finally in scission and uncoating of the nascent vesicle. In addition, many of the accessory components are involved in regulating and coupling the actin cytoskeleton to the endocytic membrane. We will discuss the different accessory components and their various roles. Most of the data comes from studies performed with cultured mammalian cells or yeast cells. The process of endocytosis is well conserved between these different organisms, but there are also many interesting differences that may shed light on the mechanistic principles of endocytosis.Receptor-mediated endocytosis is the process by which eukaryotic cells concentrate and internalize cell surface receptors from the plasma membrane into small (∼50 nm– ∼100 nm diameter) membrane vesicles (Chen et al. 2011; McMahon and Boucrot 2011; Weinberg and Drubin 2012). This mechanism has been studied extensively in mammalian tissue culture cells and in yeast, and despite the evolutionary distance between yeast and mammalian cells the mechanism of receptor-mediated endocytosis in the respective cell types show remarkable similarities. Indeed many of the ∼60 endocytic accessory proteins (EAPs) found in yeast have homologs in mammalian cells, although both cell types also have unique EAPs (McMahon and Boucrot 2011; Weinberg and Drubin 2012).In the following, we briefly describe known yeast and mammalian EAPs (Sigismund et al. 2012; see also Bökel and Brand 2013; Cosker and Segal 2014; Di Fiore and von Zastrow 2014).

Table 1.

Key endocytic proteins in mammals and in yeast

Fluorescently Labeled Methyl-Beta-Cyclodextrin Enters Intestinal Epithelial Caco-2 Cells by Fluid-Phase Endocytosis

Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10⁻⁸ cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.     

The Initiation of Clathrin-Mediated Endocytosis Is Mechanistically Highly Flexible

1. Download : Download high-res image (359KB)
2. Download : Download full-size image

     

Cell Entry of Borna Disease Virus Follows a Clathrin-Mediated Endocytosis Pathway That Requires Rab5 and Microtubules

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.Borna disease virus (BDV) causes central nervous system disease in a variety of vertebrate species that is frequently manifested by behavioral abnormalities (27, 59). BDV is the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep (21, 26, 47). However, current evidence indicates that the natural host range, prevalence, and geographic distribution of BDV are wider than originally thought (25, 31). Experimentally, BDV has a wide host range, and both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (20, 23, 34, 50). Notably, cases of proventricular dilatation disease affecting different species of psittacine birds have recently been linked to infection with avian bornaviruses (24, 29), a finding that expands the natural host range of bornavirus infections associated with clinical manifestations.BDV is an enveloped virus with a nonsegmented negative-strand RNA genome (11, 33, 53, 55) whose gene organization [3′-N-P-p10 (X)-M-G-L-5′] is characteristic of mononegaviruses. However, on the basis of its unique genetic and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales.The BDV surface glycoprotein G plays a key role in receptor recognition and cell entry (20, 46). The G gene directs the synthesis of a precursor, GPC, with a predicted M_r of ca. 56 kDa, but due to its extensive glycosylation, GPC migrates with an M_r of 84 to 94 kDa. GPC is posttranslationally cleaved by the cellular protease furin into GP-1 and GP-2, corresponding to the N-and C-terminal regions, respectively, of G (2, 8, 19, 49). GP-1 has been shown to be sufficient for virus cell entry via receptor-mediated endocytosis (46), whereas GP-2 likely mediates the pH-dependent fusion event between BDV and cell membranes required for a BDV productive infection (19). In vivo, neurons are the initial target of BDV, suggesting a restricted expression pattern of a yet-unidentified virus receptor. Late in infection, BDV is detected in many tissues and organs as a consequence of its centrifugal spread via the axoplasm of peripheral nerve tissues. Receptor-independent mechanisms also contribute to cell-to-cell propagation of BDV (8).The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of the mechanisms involved in BDV cell entry. To overcome this problem, we generated a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVΔG*/BDVG) (45). Cells infected with rVSVΔG*/BDVG produced high titers (10⁷ PFU/ml) of cell-free virus progeny. Notably, rVSVΔG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV, thus providing a unique tool for the investigation of BDV G-mediated cell entry.Viruses that enter cells via receptor-mediated endocytosis mainly use trafficking pathways mediated by either clathrin or caveola, although alternative entry pathways have been also reported (36). Nevertheless, clathrin-mediated endocytosis (CME) is the route most commonly used by enveloped viruses for cell internalization (35). The initial virus-cell surface receptor interaction results in the activation of different signaling pathways leading to the accumulation of clathrin coated-pits and subsequent formation of endocytotic vesicles (43). Another major endocytotic pathway used by several viruses, including Ebola virus (16) and SV40 (44), uses caveolae for viral internalization into the cell. This endocytotic pathway is strictly dependent on recruitment of lipid rafts to the cell surface, an event mediated by cholesterol. In this regard, we have recently documented the requirements of cholesterol and structural integrity of cell surface lipid rafts for efficient cell entry of BDV (9).In this work, we provide evidence for the first time that BDV cell entry follows a CME-dependent, caveola-independent pathway. Moreover, we show that BDV entry is Rab5 dependent but Rab7 independent and that BDV G-mediated fusion has a rapid kinetics and an optimal pH between 6.0 and 6.2. These findings indicate that BDV G-mediated fusion occurs within the early-endosome compartment. We also provide evidence that microtubules, but not actin dynamics, play a role in BDV cell entry likely by mediating trafficking of BDV-containing endosomes to the subcellular location where viral and endosomal membranes fuse.     

Hemagglutination by Rabies Virus

Goose erythrocytes were agglutinated by five strains of rabies virus grown in monolayer cell cultures at pH 6.4 and at 0 to 4 C. Hemagglutination was not affected by the cell type in which the virus was grown. Prerequisites for occurrence of hemagglutination are absence of hemagglutination inhibitors (such as those contained in bovine serum) and a relatively high virus concentration (> 10(6) plaque-forming units of virus per ml). "Soluble" hemagglutinin was not present in crude preparations of extracellular virus. Treatment of purified preparations of extracellular virus with Tween 80 and ether did not result in release of a "soluble" hemagglutinin. The hemagglutinating property of extracellular virus seemed to be conditioned by the integrity of its coat. Preparations of infectious intracellular virus exhibited about 15 times lower hemagglutinating activity than extracellular virus. This decreased hemagglutinating activity did not seem to be caused by binding of hemagglutination inhibitors to the virus particles. Rabies virus can be quantitatively adsorbed onto and eluted from erythrocytes. Erythrocytes pretreated with rabies virus retained their ability to be agglutinated by the same virus strain. The reaction with rabies virus of erythrocytes treated with the receptor-destroying enzyme or KIO(4) was the same as that of nontreated erythrocytes. The hemagglutinating component of rabies virus, therefore, does not exhibit neuraminidase activity. Treatment of extracellular virus by various agents indicated that the hemagglutinating component consists of protein or lipoprotein. Sulfhydryl groups present in the viral hemagglutinin are essential for hemagglutination.     

Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments

Background

The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells.

Methodology/Principal Findings

Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl β-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island.

Conclusions

Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.     

Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis

Rhesus rhadinovirus (RRV) is a gammaherpesvirus closely related to Kaposi''s sarcoma-associated herpesvirus (KSHV), an oncogenic virus linked to the development of Kaposi''s sarcoma and several other lymphoproliferative diseases, including primary effusion lymphoma and multicentric Castleman''s disease. RRV naturally infects rhesus macaques and induces lymphoproliferative diseases under experimental conditions, making it an excellent model for the study of KSHV. Unlike KSHV, which grows poorly in cell culture, RRV replicates efficiently in rhesus fibroblasts (RFs). In this study, we have characterized the entry pathway of RRV in RFs. Using a luciferase-expressing recombinant RRV (RRV-luciferase), we show that the infectivity of RRV is reduced by inhibitors of endosomal acidification. RRV infectivity is also reduced by inhibitors of clathrin-mediated but not caveola-mediated endocytosis, indicating that RRV enters into RFs via clathrin-mediated endocytosis. Using a red fluorescent protein (RFP)-expressing recombinant RRV (RRV-RFP), we show that RRV particles are colocalized with markers of endocytosis (early endosome antigen 1) and clathrin-mediated endocytosis (clathrin heavy chain) during entry into RFs. RRV particles are also colocalized with transferrin, which enters cells by clathrin-mediated endocytosis, but not with cholera toxin B, which enters cells by caveola-mediated endocytosis. Inhibition of clathrin-mediated endocytosis with a dominant-negative construct of EPS15, an essential component of clathrin-coated pits, blocked the entry of RRV into RFs. Together, these results indicate that RRV entry into RFs is mediated by clathrin-mediated endocytosis.Kaposi''s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is a gammaherpesvirus associated with the development of Kaposi''s sarcoma, a malignancy commonly found in AIDS patients (13). KSHV is also associated with the development of multicentric Castleman''s disease (MCD) and primary effusion lymphoma (PEL), two rare lymphoproliferative diseases. KSHV has a restricted host range, making it difficult to study KSHV and its related malignances directly in an animal model (25). Rhesus rhadinovirus (RRV) is closely related to KSHV. RRV infects its natural host and induces lymphoproliferative diseases resembling MCD and PEL; thus, it has been proposed as an animal model for the study of KSHV (19, 26, 39). Two isolates of RRV (26-95 and 17577) have been independently isolated and sequenced so far (3, 7, 32).To establish a successful infection, a virus needs to enter the target cells and release its genome (20). Thus, defining the entry and trafficking pathway of RRV can help us understand its mechanism of infection and replication in vitro and in vivo. Herpesviruses bind to the cell surface through complex interactions between viral glycoproteins and receptor molecules, leading to either plasma membrane fusion or endocytosis (35). Plasma membrane fusion is a pH-independent event between the viral envelope and the host cell plasma membrane (23). Enveloped viruses also take advantage of cellular endocytosis pathways for their internalization (34). Endocytosis leads to fusion between the membrane of the internalized vesicle and the viral envelope at low pHs and to the release of the viral particle into the cytoplasm. Following membrane fusion, the nucleocapsid traffics to the perinuclear space and delivers the viral genome to the nucleus. Thus, endocytosis offers a convenient and fast transit system enabling the virus to enter and traffic across the plasma membrane and cytoplasm of the infected cell.In mammalian cells, there are several endocytic pathways, including clathrin-mediated endocytosis, caveola-mediated endocytosis, clathrin- and caveola-independent endocytosis, and macropinocytosis (34). These endocytic pathways differ in the nature and size of the cargo. The clathrin-mediated pathway is the most commonly observed uptake pathway for viruses (30). A viral particle is internalized into a clathrin-coated vesicle, which then loses the clathrin-coated subunits before fusing with the early endosome. An activation step occurs in the endosome, leading to the fusion of the viral envelope with the endosomal membrane and the delivery of the viral capsid to the cytosol. The acidic pH in the endosome is thought to play an essential role in triggering the fusion event. Therefore, pH sensitivity is often considered an indication that a virus enters the cell by endocytosis (30).KSHV has been shown to use clathrin-mediated endocytosis to enter human foreskin fibroblasts, activated primary human B cells, and primary human umbilical vein endothelial cells (1, 12, 29); however, the macropinocytic pathway and plasma membrane fusion pathway have also been implicated (17, 28). The mechanism of RRV entry into cells has not been defined. In this study, using two recombinant RRVs expressing luciferase (RRV-luciferase) and red fluorescent protein (RRV-RFP), respectively, we have characterized the entry pathway of RRV in rhesus fibroblasts (RFs), a cell type that RRV can infect efficiently and in which it can replicate. The results show that RRV entry into RFs occurs primarily via clathrin-mediated endocytosis.     

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.     

Absorption of Proteoglycan via Clathrin-Mediated Endocytosis in the Small Intestine of Rats

An alginate was isolated from commercially cultured Nemacystus decipiens which had been harvested in Yonashiro Town (Okinawa, Japan). The yield of the alginate was 1.6% (w/w of wet alga), and the uronic acid, ash and moisture contents of the alginate were 86.0%, 12.0%, and 2.3% (w/w), respectively. The molecular mass of the alginate was estimated to be about 1.5×10⁵. The infrared spectrum and optical rotation of the alginate were in agreement with those of the standard alginate. D-Mannuronic acid and L-guluronic acid were identified by ¹H- and ¹³C-NMR spectroscopy, the molar ratio of both sugar residues being estimated to be 0.72:1.00.     

LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser⁷³⁷ in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl⁻ secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser⁷³⁷ mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl⁻ channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl⁻ channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.     

用狂犬病毒核蛋白基因小干扰RNA库抑制狂犬病毒复制和感染的研究

采用RNaseⅢ消化长片段双链RNA的方法,制备了狂犬病毒N基因、P基因和G基因小干扰RNA库(siRNACocktail)。将siRNA转染BSR和MNA细胞单层后,采用直接免疫荧光法观察发现,N基因siRNACocktail能够对RV的复制和感染产生明显和稳定的抑制作用,并且通过RT-PCR在转录水平探测到mRNA产量的降低;而P基因或G基因siRNACocktail对RV的复制和感染不产生或仅产生弱的抑制作用。这一结果为RNA干扰在RV研究中靶基因的选择及进一步的应用提供了依据。