Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.     

JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal alpha(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.     

Inhibitors of COP-mediated transport and cholera toxin action inhibit simian virus 40 infection

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20 degrees C. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.     

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin alphavbeta6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37 degrees C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin alphavbeta6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.     

Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER

Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.     

Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells

Infection of mammalian cells with Semliki Forest virus requires the endocytosis of the virus, its delivery to prelysosomal endosomes, and fusion of the viral envelope with the endosome membrane. Previous studies have indicated that the low endosomal pH triggers a conformational change in the viral spike glycoproteins rendering them fusogenic. In this paper, we demonstrate an additional factor(s) which regulates virus fusion in endosomes. We found that Semliki Forest virus is unable to penetrate or infect baby hamster kidney (BHK-21) cells grown in medium containing reduced Na+ concentrations. Virus endocytosis and degradation are nearly normal, the virus is transported to endosomes where a characteristic low pH-induced loss of trypsin-sensitivity of the E1 spike glycoprotein occurs. Nevertheless, the viral envelope fails to fuse with the endosomal membrane and the viral RNA is not released into the cytosol. As judged by the uptake of the voltage-sensitive probe [3H]triphenylmethyl phosphonium we observed a close correlation between conditions which inhibit virus infection and which cause depolarization of the cells. We propose that in intact cells, the fusion of Semliki Forest virus with the endosome membrane depends not only on acidic endosomal pH, but also on the maintenance of the potential.     

Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation.

Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40- or BKV-transformed cells.     

A model of the binding, entry, uncoating, and RNA synthesis of Semliki Forest virus in baby hamster kidney (BHK-21) cells

A quantitative understanding of viral trafficking would be useful in treating viral-mediated diseases, designing protocols for viral gene therapy, and optimizing heterologous protein production. In this article, a model for the trafficking of Semliki Forest virus and its RNA synthesis in baby hamster kidney (BHK-21) cells is presented. This model includes the various steps leading to infection such as attachment, endocytosis, and viral fusion in the endosome. The model estimates a mean fusion time of 4 to 6 min for the wild-type virus, and 38 min for Fus-1, an SFV mutant which requires a lower pH for fusion. These mean fusion times are consistent with the time-scale of endosomal acidification, suggesting viruses fuse almost instantaneously with the endosomal membrane as soon as the pH of the endosome drops below the pH threshold of the virus. Infection is most likely controlled at the level of viral uncoating, as shown by the close agreement between the efficiency of uncoating and the experimentally determined fraction of viruses that is infectious. The viral RNA synthesized per cell is best described by assuming that it depends on the number of uncoated viruses prior to the onset of replication according to a saturation-type expression. A Poisson distribution is used to determine the distribution of uncoated viruses among the cells. Because attachment is the rate-limiting step in the uncoating of the virus, increasing the attachment rate can lead to enhanced RNA synthesis and, hence, new virion production. Such an increase in the attachment rate may be obtained by lowering the medium pH or the addition of a polycation. (c) 1995 John Wiley & Sons, Inc.     

Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles

Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains beta-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some beta-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.     

Effect of Bafilomycin A1 and Nocodazole on Endocytic Transport in HeLa Cells: Implications for Viral Uncoating and Infection

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used ³⁵S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.     

A JC virus-induced signal is required for infection of glial cells by a clathrin- and eps15-dependent pathway

Infectious entry of JC virus (JCV) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. In this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. Transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. We also show that the JCV-induced signal activates the mitogen-activated protein kinases ERK1 and ERK2. These data demonstrate that JC virus binding to human glial cells induces an intracellular signal that is critical for entry and infection by a ligand-inducible clathrin-dependent mechanism.     

A plant RNA virus hijacks endocytic proteins to establish its infection in plants

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor‐mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin‐related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co‐opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV‐infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1‐dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.     

Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.     

Identification of critical elements within the JC virus DNA replication origin.

The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.     

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.     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

Mapping 5'' termini of JC virus early RNAs.

Within its enhancer promoter region, the MAD-1 strain of JC virus (JCV) has two 98-base-pair tandem repeats, each containing a TATA box-like sequence. In the present study, polyadenylated early JCV mRNAs were isolated 5 or 29 days after infection of primary human fetal glial (PHFG) cells. By using S1 nuclease, the 5' termini of the early mRNAs were mapped to nucleotide position(s) (np) 122 through 125, which lies within an AT rich region (at np 113 through 127). In contrast, when JCV DNA was transcribed in vitro, we observed a single major cluster of 5' start sites at np 94 through 97, which is approximately 25 base pairs downstream from one of the TATA boxes. By day 5, the earliest time at which JCV RNA was detected, viral DNA replication had begun; it continued for at least an additional 20 days. Since more late than early RNA was present at 5 days postinfection, the early RNAs whose synthesis began at np 122 through 125 may be analogous to SV40 late early mRNA (Ghosh and Lebowitz, J. Virol. 40:224-240, 1981). However, we have not detected RNAs with 5' termini 25 to 30 bp downstream from the TATA box at earlier times. While JCV contains two identical TATA boxes, one in each of the 98-bp repeats, only the upstream TATA box functions as an early promoter element.     

Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR

Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.