Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection

The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.     

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

The Initiation of Clathrin-Mediated Endocytosis Is Mechanistically Highly Flexible

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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.     

High-Affinity Dkk1 Receptor Kremen1 Is Internalized by Clathrin-Mediated Endocytosis

Kremens are high-affinity receptors for Dickkopf 1 (Dkk1) and regulate the Wnt/β-catenin signaling pathway by down-regulating the low-density lipoprotein receptor-related protein 6 (LRP6). Dkk1 competes with Wnt for binding to LRP6; binding of Dkk1 inhibits canonical signaling through formation of a ternary complex with Kremen. The majority of down-regulated clathrin-mediated endocytic receptors contain short conserved regions that recognize tyrosine or dileucine sorting motifs. In this study, we found that Kremen1 is internalized from the cell surface in a clathrin-dependent manner. Kremen1 contains an atypical dileucine motif with the sequence DXXXLV. Mutation of LV to AA in this motif blocked Kremen1 internalization; as reported previously for other proteins, the aspartic acid residue in Kremen1 is not critical. Inhibition of expression of the adaptor protein 2 (AP-2) or inhibition of clathrin by pitstop 2 also blocked Kremen1 internalization. The novel amino acid sequence identified in Kremen1 is similar to the motif previously identified in hydra, yeast, and other organisms known to signal from the trans-Golgi network to the endosomal compartment.     

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.     

Different Rotavirus Strains Enter MA104 Cells through Different Endocytic Pathways: the Role of Clathrin-Mediated Endocytosis

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins α2β1 and αvβ3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.Endocytosis is a cellular process that involves the formation of a vesicle whose cargo is transported from the extracellular milieu to the interior of the cell. Several endocytic pathways have been described, and all of them have been shown to be used by viruses during cell entry. These pathways include clathrin-mediated endocytosis, uptake via caveolae, macropinocytosis, phagocytosis, and a novel non-clathrin-, non-caveola-mediated pathway that is currently not well characterized (32). While detailed information about the entry of several enveloped viruses is now available (4, 35, 49, 53, 56), the mechanism by which nonenveloped viruses enter cells is not well understood. Two general mechanisms have been proposed to be used by these viruses to reach the cell''s cytoplasm: direct penetration at the cell surface, during which the viral particles are directly translocated from the external milieu into the cytoplasm, or internalization through endocytic processes (55).Rotaviruses, members of the family Reoviridae, are the leading etiologic agent of viral gastroenteritis in infants and young children worldwide, being responsible for an estimated 500,000 deaths each year (41). These nonenveloped viruses are formed by three concentric layers of protein that surround the viral genome, formed by 11 segments of double-stranded RNA. The outermost layer of the virion is formed by two proteins, VP4 and VP7, which are involved in the early interactions of the virus with its host cell (7, 27). VP4 is involved in receptor binding and cell penetration. The role of VP7 is less clear, although it has been shown that it interacts with the cell surface molecules at a postattachment step (17). After binding to the cell surface, the virus penetrates the plasma membrane to productively infect the cell. This penetration depends on the trypsin treatment of the virus, which results in the specific cleavage of VP4 to polypeptides VP8 and VP5. This cleavage promotes VP4 rearrangements in the viral particles that rigidify the spikes (7, 11).Despite the fact that, in vivo, rotaviruses primarily infect the mature enterocytes of the small intestine, studies of the infection of this type of cells have been limited due to the lack of established intestinal cell lines of small intestine origin. Given the absence of a better model, most of the studies on the entry and replication cycle of rotavirus have been conducted either in the epithelial monkey kidney cell line MA104 or in the human colon carcinoma cell line Caco-2, which are highly permissible to these viruses.Using as a model MA104 cells and the simian rotavirus RRV, we have proposed that rotavirus cell entry is a complex multistep process that involves the two virus surface proteins and several cell receptors, including sialic acids, gangliosides, integrins α2β1, α4β1, αvβ3, and αxβ2, and the heat shock cognate protein hsc70 (22). We have also shown that depletion of cholesterol from the cellular membrane severely impairs the infectivity of rotavirus (19, 50) and have suggested that sphingolipid- and cholesterol-enriched membrane lipid microdomains might be involved in rotavirus cell entry, since the virus and its receptors associate with these domains at early times during infection (23). However, there are some rotavirus strains that may not use all these molecules; some rotavirus strains are resistant to the neuraminidase (NA) treatment of the cell, and thus, they have been classified as NA resistant (6, 34). Additionally, the infectivity of some viral strains is not blocked by anti-integrin antibodies, suggesting the existence of rotavirus strains that are integrin independent (Table ​(Table1)1) (17).

TABLE 1.

Cellular receptor requirements of different rotavirus strains

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.     

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.     

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.