Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection. Received: 11 October 1996     

Mechanism of entry of human rhinovirus 2 into HeLa cells

Internalized human rhinovirus 2 (HRV2) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20 degrees but not at 34 degrees the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20 degrees prior to addition of monensin permits virus synthesis to occur, depending on the time of preincubation.     

Entry of human rhinovirus 89 via ICAM-1 into HeLa epithelial cells is inhibited by actin skeleton disruption and by bafilomycin

HRV89, a major-group rhinovirus, uses intercellular adhesion molecule 1 (ICAM-1) for cell entry, while minor-group HRV2 uses the LDL receptor for clathrin-mediated endocytosis. Entry of HRV89 into HeLa epithelial cells was found to be inefficient, and infectious virus was still detected on the plasma membrane after 3 h of incubation with the cells. Endocytosis, and consequently infection, of HRV89 but not of HRV2, was almost completely blocked by the actin-polymerization inhibitor cytochalasin D, while the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on infection with either virus. Cytochalasin D also inhibited major-group HRV infection of rhabdomyosarcoma cells expressing ICAM-1 when the time available for uncoating was limited to 30 min. Although cholesterol depletion strongly inhibited HRV89 infection of HeLa cells, it only slightly affected HRV89 endocytosis, indicating that a lipid raft environment was not essential for virus uptake. The sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) significantly reduced cell entry and infection by HRV89 only at a concentration that also inhibited HRV2 infection and Alexa 488-transferrin entry. These data rule out classical macropinocytosis as an infectious entry pathway of HRV89 in HeLa cells. Notably, the proton ATPase inhibitor bafilomycin strongly affected cell entry of both viruses, suggesting a role for submembraneous pH in rhinovirus endocytosis.     

Release of canine parvovirus from endocytic vesicles

Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles.     

Ionic requirements for entry of Shiga toxin from Shigella dysenteriae 1 into cells

The ionic requirements for entry of Shiga toxin into cells were examined by measuring inhibition of protein synthesis after short-term incubations with toxin. The sensitivity of Vero cells and HeLa cells to Shiga toxin was strongly dependent on the divalent cation present. Vero cells were most sensitive in the presence of CaCl2 and SrCl2, whereas HeLa cells were equally sensitive in the presence of MgCl2, SrCl2, and CaCl2. Both cell lines were protected by BaCl2, CoCl2, and MnCl2. Inhibitors of Ca2+ transport, like verapamil, D600, and Co2+ as well as the calcium-ionophores A23187 and ionomycin, protected both cell lines. HEp-2 cells were protected against Shiga toxin by a high concentration of potassium in the medium as well as by potassium depletion of the cells. Substitution of chloride in the medium with slowly permeable anions, like SO42- and SCN-, protected the cells against Shiga toxin. High concentrations of the ionophore nigericin that increase pH of acidic intracellular vesicles did not protect Vero cells against Shiga toxin. Shiga Toxin X-114 at pH values below 4.5. This binding was shifted to higher pH values after pretreatment of the toxin with dithiothreitol. The results indicate that Ca2+ transport through physiologically occurring Ca2+ channels is required for entry of Shiga toxin into cells. Furthermore, the sensitivity of cells of Shiga toxin is strongly dependent on the anions present.     

Entry of Shigella dysenteriae toxin into HeLa cells

The rate of shigella toxin entry into the cytosol of HeLa S3 cells was estimated from the toxin-induced reduction in protein synthesis. Whereas high toxin concentrations strongly reduced protein synthesis within 30 min, lower concentrations required longer times. The major part of the cell-bound toxin entered only after several hours. Toxin entered cells after incubation at 25 degrees C but not at 20 degrees C, although toxin binding was the same at the two temperatures. Increasing the KCl concentration to 0.2 M protected against toxin. The toxin entry was strongly reduced when the level of ATP in the cells was reduced by incubation with metabolic inhibitors. Lysosomotrophic agents such as NH4Cl and chloroquine had little or no protective effect, but the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone and the ionophore monensin protected cells against the toxin. Cells were also protected when the pH was reduced to 6.4. The entry of shigella toxin is discussed in relation to that of other protein toxins with intracellular sites of action.     

Evidence for endocytosis-independent infection by human rotavirus

Summary The effects of five lysosomotropic drugs (NH₄Cl, chloroquine, methylamine, amantadine and dansylcadaverine) and cytochalasin B on human rotavirus (HRV KUN strain) and vesicular stomatitis virus (VSV Indiana strain) infection in monkey MA 104 cells were examined. These drugs had little effect on HRV yield but greatly reduced VSV yield. The results strongly suggest that HRV does not require endocytotic activity and intracellular acidic vesicles for the initial stage of infection and support our postulate that HRV enters the target cell by direct penetration of its nucleoid through the cell membrane.     

Biochemical studies on the entry of sindbis virus into BHK-21 cells and the effect of NH4Cl

The importance of a lysosomal pathway for the infection of cells by Sindbis virus was studied. BHK-21 cells were infected with Sindbis virus radiolabeled with l-[³⁵S]methionine and the fate of viral proteins was followed up to 120 min postinfection. Virus was transiently associated with a subcellular fraction enriched in lysosomes (identified by four-to to efold enrichment in the enzyme marker acid phosphatase). Capsid proteins were preferentially released into the cytoplasm after 60 min of infection and significant degradation of viral proteins occurred between 1 and 2 hr after infection. NH₄Cl, a weak base thought to increase the intralysosomal pH and impair lysosomal function, blocked an early event between the binding of virus on the cell surface and penetration into lysosomes. This contradicts the presumed lysosomal site of action of this chemical on Sindbis virus infection and is consistent with an effect on receptor clustering into coated pits or the delivery of endocytic vesicle content to lysosomes. The results of these studies indicate the involvement of intracellular vesicles, either lysosomes or vesicles of similar size, and thus a receptor-mediated endocytic pathway (viropexis) as the primary mechanism for entry of Sindbis virus into BHK-21 cells.     

Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells

Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment.     

Investigation of the anti-viral mechanism of poly I and poly C against encephalomyocarditis virus infection in the absence of interferon induction in mice.

Protection of mice against EMC virus infection by poly C and poly I has already been distinguished from interferon mediated protection in several ways. Transfer of serum from EMC virus infected and poly C or poly I treated mice to donor mice that were then infected shows that the anti-viral effect of the single-stranded polynucleotides is not due to boosting interferon produced by infection itself in the way that inferferon can be 'primed' in vitro. Mice surviving infections of more than I X LD100 as a result of poly C or poly I treatment show no protection against re-infection 15 days after the first infection, indicating no long-term stimulation of immune responses to the virus. Mice treated with an immunosuppressive regime of cytosine arabinoside can be protected against EMC virus infection with poly C and poly I treatment and athymic 'nude' mice can also be protected. The possibility of IgM stimulation by poly C and poly I seems unlikely from experiments in which serum was transferred from mice treated with the polynucleotides and an inactivated EMC 'vaccine' to recipient mice which were then challenged with infectious virus. Protection of mice against EMC virus by the single-stranded polynucleotides is abolished by administration of silica to the mice, implying an involvement of macrophages in the protective effects of poly C and poly I. The possibility that the polynucleotides stimulate clearance of virus particles, at least from immunologically responsive regions of the mouse, has been discounted by the inability of polynucleotide treatment to suppress 'vaccine' mediated protection of mice. These results indicate that macrophages are involved in the anti-viral effects of poly C and poly I either because they inhibit replication of the virus in macrophages or because direct anti-viral properties of macrophages are activated by the polynucleotides.     

Modification of membrane permeability induced by animal viruses early in infection

The infection of mouse L cells by encephalomyocarditis (EMC) virus renders the cell permeable to several translation inhibitors to which normal cells are impermeable. The modification of membrane permeability to the aminoglycoside antibiotic hygromycin B takes place early in infection during virus adsorption and also at the time when large amounts of viral coat proteins are synthesized. The specific inhibition of protein synthesis in virus-infected cells early in infection is also observed with other translation inhibitors such as anthelmycin, gougerotin, and edeine, indicating that a general permeabilization takes place during virus adsorption. The presence of 1 mM hygromycin B in the culture medium during the first hour of EMC infection strongly reduced the production of new infectious virus. In order to understand the molecular mechanisms involved in membrane permeabilization early during EMC infection, the cells were treated with compounds known to disrupt microtubules and microfilaments and to block pinocytotic processes, e.g., vinblastine, cytochalasin B, and colchicine. However, even in the presence of those compounds the early leakiness phenomenon was still observed. The presence of concanavalin A, which binds to certain cell surface recptors, or inhibitors that block energy production (e.g., FCCP and NaNs) had no significant effects on the modification of membrane permeability. Treatment of human cells with interferon does not prevent the increase in membrane permeability after EMC infection. No viral or cellular protein synthesis is necessary for the early membrane leakiness to occur, suggesting that virions themselves are responsible for this modification. The alteration of permeability to translation inhibitors is also observed after the infection of different mammalian cell lines with different viruses such as vesicular stomatitis virus and Semliki Forest virus, two lipid-enveloped RNA-containing viruses.     

Effect of lysosomotropic compounds on early events in foot-and-mouth disease virus replication

The effect of three lysosomotropic compounds, chloroquine, monensin and NH4Cl, on the replication of foot-and-mouth disease virus (FMDV) type A12 was studied. Viral replication was almost totally inhibited by 0.5 mM chloroquine, 50 microM monensin, or 25 mM NH4Cl. Monensin and NH4Cl affected replication when added either before or within the first hour of infection. Chloroquine, however, still inhibited viral replication when added up to 2.5 h after infection. Assays of binding of radiolabeled virus to cells showed that these compounds had no effect on viral adsorption. Neither monensin nor NH4Cl had any significant effect on cellular protein synthesis, but there was no evidence of viral protein synthesis in cells infected in the presence of these compounds. In contrast, chloroquine inhibited both cellular and viral protein synthesis. Eclipse assays, performed in the presence of the compounds, showed that while chloroquine and NH4Cl had little effect on cell-induced degradation of incoming virions to 12 S protein subunits, monensin inhibited this reaction. The replication of representative members of all seven serotypes of FMDV was inhibited by monensin although some types were less sensitive to the compound than others. These results are consistent with a model which postulates that viral eclipse is the result of acidification of endocytic vesicles which degrade entrapped virions to 12 S protein subunits resulting in the release of genome RNA.     

Effect of ammonium 5-tungsto-2-antimoniate on encephalomyocarditis and vesicular stomatitis virus infections in mice.

Ammonium 5-tungsto-2-antimoniate (HPA 23) protected micr partially or completely against two strains of encephalomyocarditis (EMC) virus and one strain of vesicular stomatitis (VSV) virus. The best protective effect was obtained with EMC strain VR 129 and VSV when a single i.p. injection of HPA 23 was administered shortly before virus inoculation. Mice protected by HPA 23 against EMC strain VR129 had virus titres in the blood and brain similar to those in untreated mice. A synergism between interferon and HPA 23 was observed in mice infected with EMC VR129. Our results demonstrate the in vivo activity of HPA 23 against two lethal viral infections and suggest that, at least in mice infected with EMC, death may not be related solely to virus multiplication.     

Entry of a heparan sulphate-binding HRV8 variant strictly depends on dynamin but not on clathrin, caveolin, and flotillin

The major group human rhinovirus type 8 can enter cells via heparan sulphate. When internalized into ICAM-1 negative rhabdomyosarcoma (RD) cells, HRV8 accumulated in the cells but caused CPE only after 3 days when used at high MOI. Adaptation by three blind passages alternating between RD and HeLa cells resulted in variant HRV8v with decreased stability at acidic pH allowing for productive infection in the absence of ICAM-1. HRV8v produced CPE at 10 times lower MOI within 1 day. Confocal fluorescence microscopy colocalization and the use of pharmacological and dominant negative inhibitors revealed that viral uptake is clathrin, caveolin, and flotillin independent. However, it is blocked by dynasore, amiloride, and EIPA. Furthermore, HRV8v induced FITC-dextran uptake and colocalized with this fluid phase marker. Except for the complete inhibition by dynasore, the entry pathway of HRV8v via HS is similar to that of HRV14 in RD cells that overexpress ICAM-1.     

Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1.

Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.     

Mechanism of astrovirus entry into Graham 293 cells.

Astroviruses are intestinal pathogens associated with gastroenteritis in man and animals. The mechanism of internalization into host cells has not been reported previously. The cell entry pathway of serotype 1 human astrovirus into 293 cell line was studied biochemically and morphologically. Viral infection was monitored by indirect immunofluorescence. Infected cells were treated with the lysosomotropic agents ammonium chloride, methylamine, and dansylcadaverine or the ionophore monensin to raise the intraendosomal and intralysosomal pH. All drugs tested inhibited the early stages of infection whereas they did not interfere with the viral binding to the plasma membrane. The presence of astrovirus particles was detected by electron microscopy in coated pits and later in coated vesicles. The data indicate adsorptive endocytosis as the most probable mechanism by which astroviruses enter susceptible cells.     

Vaccinia-mediated rescue of encephalomyocarditis virus from the inhibitory effects of interferon

Coinfection of mouse L cells with vaccinia virus rescues encephalomyocarditis virus (EMC) from the inhibitory effect of interferon (IFN). The vaccinia-mediated rescue of EMC growth increases the yield of EMC as much as 1000-fold and is optimum when vaccinia is used at a multiplicity of infection of 1. This rescue correlates with a vaccinia-dependent stimulation of EMC gene expression. Evidence is presented to indicate that the rescue by vaccinia does not involve a block of the 2'-5'A synthetase pathway. However, the vaccinia rescue function is correlated with a vaccinia-mediated inhibition of the IFN-induced protein kinase.     

Vesicles containing Chlamydia trachomatis serovar L2 remain above pH 6 within HEC-1B cells.

Chlamydia trachomatis serovar L2 is an obligate intracellular bacterium which is internalized in target epithelial cells by endocytosis and resides within a membrane-bound vesicle. Over the next several hours following entry, individual serovar L2-containing vesicles fuse with one another to form a single membrane-bound vesicle (or inclusion) within which the microcolony develops. The experiments reported here directly examined the pH of vesicles containing chlamydiae. The pH was determined by measuring emission ratios of the fluorescent, pH-sensitive probe SNAFL (5-[and 6-]-carboxyseminaphthofluorescein-1, succinimidyl ester) conjugated to chlamydiae. The pH remained above 6.0 at 2, 4, and 12 h after infection, while the pH of vesicles contained heat-killed organisms fell 5.3. In the presence of amines, which raise the pH of acidic compartments, C. trachomatis inclusion formation was unaffected. Inactivation of Na+,K+ -ATPases, the ion pumps responsible for maintaining a pH above 6 within early endocytic vesicles, inhibited the growth of C. trachomatis within epithelial cells. Preventing vesicular acidification by inhibiting the vacuolar proton ATPase did not affect chlamydial growth. Thus, chlamydiae do not reside within highly acidic vesicles and avoid the pathway leading to lysosomes.     

Inhibition of Dengue Virus Entry into Target Cells Using Synthetic Antiviral Peptides

Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.