Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway

The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.     

Effect of a capsid-stabilizing pyridazinamine, R 78206, on the eclipse and intracellular location of poliovirus.

R 78206 (a pyridazinamine derivative) inhibits the formation of poliovirus eclipse particles. Its effect on the intracellular location of poliovirus was studied by separating subcellular fractions in iso-osmotic Nycodenz gradients. The compound did not inhibit internalization of intact virus into small lipid vesicles, but it did inhibit the release of virus from these vesicles and its entry into lysosomes.     

Morphological study on the entry of African swine fever virus into cells

The early interactions between African swine fever virus (ASFV) and monkey kidney cells in culture, and the effect of chloroquine were studied by electron microscopy. Our results indicate that ASFV uptake occurs by endocytosis: after attachment to the cell surface, the virions were seen in coated pits and were internalized by endocytosis in endosomes and finally in lysosomes. Virions in coated vesicles were never seen. All these steps were completed in about 15 min. Direct penetration of viruses through the plasma membrane was never observed. In order to elucidate the participation of an acidic intracellular compartment in the penetration of ASFV, we studied the effect of chloroquine, a lysosomotropic drug known to increase the pH of acidic intracellular vacuoles and to inhibit ASFV infection. In the presence of this drug there were no apparent alterations on binding, endocytosis and intracellular distribution of the viral particles. The main effect of chloroquine was to retain the virions in lysosomes. When the drug was removed from the medium, the viral particles disappeared and images of binding of viral membranes with the membranes of the intracellular vacuoles were obtained, suggesting that the inhibited step is the uncoating of the virus. Viral fusion with the plasma membrane was obtained when the medium was acidified to pH 5-6. These results suggest that ASFV enters the cells by adsorptive endocytosis and that the uncoating process takes place intracellularly in a way similar to that described for Semliki Forest virus and other enveloped viruses.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.     

Ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection

Acid sphingomyelinase (ASMase) converts the lipid sphingomyelin (SM) to phosphocholine and ceramide and has optimum activity at acidic pH. Normally, ASMase is located in lysosomes and endosomes, but membrane damage or the interaction with some bacterial and viral pathogens can trigger its recruitment to the plasma membrane. Rhinovirus and measles viruses each require ASMase activity during early stages of infection. Both sphingomyelin and ceramide are important components of lipid rafts and are potent signaling molecules. Each plays roles in mediating macropinocytosis, which has been shown to be important for ebolavirus (EBOV) infection. Here, we investigated the role of ASMase and its substrate, SM, in EBOV infection. The work was performed at biosafety level 4 with wild-type virus with specificity and mechanistic analysis performed using virus pseudotypes and virus-like particles. We found that virus particles strongly associate with the SM-rich regions of the cell membrane and depletion of SM reduces EBOV infection. ASM-specific drugs and multiple small interfering RNAs strongly inhibit the infection by EBOV and EBOV glycoprotein pseudotyped viruses but not by the pseudotypes bearing the glycoprotein of vesicular stomatitis virus. Interestingly, the binding of virus-like particles to cells is strongly associated with surface-localized ASMase as well as SM-enriched sites. Our work suggests that ASMase activity and SM presence are necessary for efficient infection of cells by EBOV. The inhibition of this pathway may provide new avenues for drug treatment.     

Heat shock cognate protein 70 is involved in rotavirus cell entry

In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.     

THE PENETRATION OF REOVIRUS RNA AND INITIATION OF ITS GENETIC FUNCTION IN L-STRAIN FIBROBLASTS

Reovirus type 3 is phagocytized by L cells and rapidly sequestered inside lysosomes. Hydrolases within these organelles are capable of stripping the viral coat proteins, but they fail to degrade the double-stranded RNA genome. These observations support the view that sojourn of reovirus in lysosomes, when the lytic enzymes uncoat its genome, is an obligatory step in the sequence of infection. Although the mechanism for transferring the uncoated RNA out of lysosomes remains to be elucidated, evidence is presented suggesting that progeny genomes are bound to site(s) possessing the fine structure of viral inclusions or factories. It appears that both the synthesis of single- and double-stranded viral RNA and the morphogenesis of progeny virus particles occur in such factories.     

Uncoating of influenza virus in endosomes

The intracellular uncoating site of influenza virus was studied by measuring the fluorescence intensity of probes conjugated to the virus or the isolated hemagglutinin and also by assaying virus replication under various incubation conditions. Acidification of the viral environment was monitored by the decrease in the fluorescence intensity of fluorescein isothiocyanate, and transport of the virus particles into secondary lysosomes was assayed by the increase in the fluorescence intensity of fluorescein isothiocyanate diphosphate. The intracellular pH was estimated by the ratio of fluorescence intensities excited at two different wavelengths. It was found that the viral environment became acidified to a pH value of 5.1 to 5.2 within 10 min at 37 degrees C or 1 h at 20 degrees C after endocytosis. Addition of ammonium chloride to the medium rapidly raised the pH to 6.7. Transport of the virus particles into the secondary lysosomes was slower and negligibly low during those incubation periods. Virus replication occurred when the cells were incubated for 10 min at 37 degrees C or for 1 h at 20 degrees C, followed by incubation in the presence of ammonium chloride for a total of 12 h. These results indicate the uncoating of influenza virus in endosomes before reaching the secondary lysosomes.     

Penetration of Semliki Forest virus from acidic prelysosomal vacuoles

To identify and characterize the intracellular site from which the penetration of Semliki Forest virus (SFV) to the cytosolic compartment of the host cell occurs, we determined the time course and temperature dependence of nucleocapsid uncoating and infection in BHK-21 cells. At 37 degrees C the genome release to the cytosol was detected within 5-7 min after virus endocytosis, whereas delivery of the virus particles to secondary lysosomes occurred within 15-20 min. At temperatures of 15 degrees -20 degrees C virus particles were internalized by endocytosis, but they were not delivered to the secondary lysosomes. Nevertheless, at 20 degrees C nucleocapsid uncoating and infection occurred, indicating that secondary lysosomes are not required for SFV penetration. We conclude that the penetration reaction occurs in prelysosomal endocytic vacuoles (endosomes). As SFV penetration by membrane fusion requires a pH less than 6 and the presence of cholesterol in the target membrane, the data indicate that endosomes are acidic and contain cholesterol.     

Mechanism of adenovirus neutralization by Human alpha-defensins

Defensins are naturally occurring antimicrobial peptides that disrupt bacterial membranes and prevent bacterial invasion of the host. Emerging studies indicate that certain defensins also block virus infection; however, the mechanism(s) involved are poorly understood. We demonstrate that human alpha-defensins inhibit adenovirus infection at low micromolar concentrations, and this requires direct association of the defensin with the virus. Moreover, defensins inhibit virus disassembly at the vertex region, thereby restricting the release of an internal capsid protein, pVI, which is required for endosomal membrane penetration during cell entry. As a consequence, defensins hamper the release of adenovirus particles from endocytic vesicles, resulting in virion accumulation in early endosomes and lysosomes. Thus, defensins possess remarkably distinct modes of activity against bacteria and viruses, and their function may provide insights for the development of new antiviral strategies.     

Retroviral retargeting by envelopes expressing an N-terminal binding domain.

We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment.     

Initial Binding of Murine Leukemia Virus Particles to Cells Does Not Require Specific Env-Receptor Interaction

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. III. Cytochemical localization of lysosomal enzymes in infected cells.

Cytochemical localization of the lysosomal enzymes acid phosphatase and arylsulfatase in cells infected by herpes simplex virus (HSV) or human cytomegalovirus (CMV) showed the following interactions between viruses and host cell lysosomes: (i) many enveloped progeny viruses were located within cytoplasmic vacuoles containing lysosomal enzyme activity; (ii) naked cytoplasmic capsids appeared to acquire an envelope by budding directly into lysosomes; and (iii) many of the cytoplasmic dense bodies that are characteristic of CMV-infected cells and are thought to represent noninfectious aggregates of CMV structural proteins (I. Sarov and I. Abady, Virology 66:464-473, 1975) also acquired a limiting membrane by budding into lysosomes. Autophagy of other cytoplasmic elements was not observed, suggesting that there is some specificity involved in the association of viral particles and CMV dense bodies with lysosomes. Despite the presence of potentially destructive hydrolases, there was little evidence of significant morphological damage to intralysosomal viruses, and high titers of infectious particles were released into the medium. It would therefore appear that significant levels of HSV and CMV infectivity normally persist even though many progeny particles are directly exposed to lysosomal enzymes.     

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.     

Electron Microscopy of Bursa of Fabricius of Chicks Infected with a Field Strain of Infectious Bursal Disease Virus

Chicks were infected in the bursa with a field strain of infectious bursal disease virus. Inter- and intracellular edema, condensation and margination of nuclear chromatin, increased number of lysosomes in macrophages, and lymphocytolytic changes appeared earliest by 8 hours post infection. Inclusions containing spheroid to hexagonal virus particles were seen in the cytoplasm of the macrophages. Multiplying virus particles in crystalline arrays arranged either in single or in multiple clusters were seen in the cytoplasm of macrophages, lymphocytes and light stained reticular epithelial cells.     

Replication of dengue viruses in mosquito cell cultures--a model from ultrastructural observations.

Mosquito cell cultures infected with human sera from dengue-1 and dengue-2 outbreaks, started in Rio de Janerio by 1986 and 1990 respectively, were examined by electron microscopy at different times post the infection of cell cultures. More information was obtained about cell penetration of virus particles in the presence or not of antibodies, their pathway inside the cells, replication mode and exist. Infectiveness of the virus at those different stages can only be attributed to the particles appearing inside the trans-Golgi vesicles; most of all newly formed virus particles remain inside the RER-derived cell vesicles or inside lysosomes, even during cell lysis. Groups of larger particles, 65-75 nm in diameter at dengue-2 infections, persist during cell passage. The large amounts of smooth membrane structures, as vesicles or tubules inside the RER, are attributed to a cell response to viral infection.     

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV)

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.     

The small chemical vacuolin-1 alters the morphology of lysosomes without inhibiting Ca2+-regulated exocytosis

Ca2+-regulated exocytosis of lysosomes was previously shown to be required for the repair of plasma membrane wounds. The small chemical vacuolin-1 alters the morphology of lysosomes without affecting the ability of cells to reseal their plasma membrane after injury. On the basis of a failure to detect Ca2+-triggered lysosomal exocytosis in vacuolin-1-treated cells, a recent study proposed that lysosomes are dispensable for resealing. Here, we show that vacuolin-1, despite altering lysosome morphology, does not inhibit the exocytosis of lysosomes induced by exposure to a Ca2+ ionophore, or by plasma membrane wounding. Thus, lysosomes cannot be excluded as agents of membrane repair in vacuolin-1-treated cells.     

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.