1 JOURNAL OF VIROLOGY, May 1991, P X/91/ $2./ Copyright 1991, American Society for Microbiology Vol. 65, No. 5 Inhibitors of Poliovirus Uncoating fficiently Block the arly Membrane Permeabiliation Induced by Virus Particles MARIA JSUIS ALMLA, MARIA UGNIA GONZALZ, AND LUIS CARRASCO* Centro de Biologia Molecular, UAM-CSIC, Universidad Aut6noma, Canto Blanco, 2849 Madrid, Spain Received 6 September 199/Accepted 18 January 1991 The entry of animal viruses into cells is associated with permeabiliation of the infected cells to protein toxins such as alpha-sarcin (C. Fernande-Puentes and L. Carrasco, Cell 2: , 198). This phenomenon has been referred to as "the early permeabiliation by animal viruses" (L. Carrasco, Virology 113: , 1981). A number of inhibitors of poliovirus growth such as WIN (3,4-dichlorophenoxy)-3-(ethylthio)-2- pyridincarbonitrile (DPC) and Ro 9-41 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabiliation induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabiliation of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalied along with heparin in endosomes in the presence of WIN 51711, DPC, or Ro We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered. The infection of animal cells by viruses is initiated by the viral recognition of specific cellular receptors (8). After attachment, the virus enters the cell, usually by an endocytotic process (22), in a manner similar to that used by other biological molecules such as hormones or growth factors (17). Once the virus has been internalied within the cell, the virus must escape the endocytotic vesicle to locate its genome either in the cytoplasm or in the nucleus (7, 11). For enveloped viruses, the viral genome is released from endosomes by fusion of the viral membrane with the endosomal membrane (7, 22). Much less is known about the processes employed by nonenveloped viruses such as poliovirus in traversing the membrane barrier of the endosome in order to liberate the genomic RNA into the cytoplasm (6, 7, 22). A number of agents that inhibit picornavirus replication bind to virion particles, preventing virus uncoating without affecting the attachment of virions to cells or their internaliation (1, 29). The detailed knowledge of some picornavirus particles at the atomic level (1, 23, 26, 3) allowed some researchers to carry out very refined studies on the interaction of WIN compounds with human rhinovirus 14 particles (2, 31, 34). These are synthetic compounds that bind to a hydrophobic pocket located within VP1 and below the floor of the 'canyon,' a deep surface depression present in these particles (2, 31, 34). This leads to a conformational change in the residues lining the canyon in human rhinovirus type 14, thus inhibiting attachment in some rhinoviruses (27). The filling of this space by the WIN compounds within the pocket also enhances protein stability and in turn inhibits uncoating in all rhinovirus particles (2, 31, 34). Several years ago, we found that animal viruses, enveloped or naked, efficiently promoted the entry of proteins into infected cells along with the virions themselves (4, 5, 13). Several protein toxins, including alpha-sarcin (13), enymes, e.g., luciferase and horseradish peroxidase (28), and other low-molecular-weight inhibitors, e.g., hygromycin B (4), can enter cells together with virion particles and are then liber- * Corresponding author ated into the cytoplasm (6). No viral gene expression was required for this early membrane permeabiliation to occur, indicating that the virion particles themselves were sufficient to promote this increased permeability (13). ven UVinactivated virions showed this effect (4, 5). These findings led us to suggest that virions lyse endosomes, thereby delivering the entrapped molecules into the cytoplasm (6). lectron microscopy studies on the permeabiliation of cells to Pseudomonas toxin caused by adenovirions also favored this hypothesis (14). Direct early permeabiliation of the plasma membrane by adenoviruses is also achieved if the culture medium is acidified, indicating that the low ph present in endosomes, together with virion particles might trigger the disruption of the endosomal membrane by naked viruses (32). As yet, no selective inhibitors of this early membrane modification induced by animal viruses are known. We now describe how compounds that block poliovirus uncoating, such as WIN (15, 29), 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DPC) (18), and Ro 9-41 (24) interfere with early permeabiliation of cell membranes, suggesting that this phenomenon is tightly coupled to virion uncoating. MATRIALS AND MTHODS Cells and viruses. Human HeLa cells were grown in Dulbecco modified agle medium supplemented with 1% newborn calf serum and containing antibiotics (1, IU of penicillin and 5 mg of streptomycin per ml). Poliovirus type 1 (Mahoney strain) was grown on HeLa cells. The virus concentration was estimated by plaque assays on the same cell line. Source of compounds. DPC was a generous gift from M. T. Kenny (Merrell Dow Research Institute, Indianapolis, Ind.). Ro 9-41 (4'-ethoxy-2'-hydroxy-4,6'-dimethoxychalcone) and Ro (4',5-dihydroxy-3-3'-7-trimethoxyflavone) were kindly given to us by H. Ishitsuka and K. Yokose (Nippon Roche Research Center, Kanagawa, Japan). WIN (5-[7-[4-[4,5-dihydro-2-oxaoly]phenoxy]heptyl]- 3-methylisoxaole) was generously provided by D. Pevear
2 VOL. 65, 1991 VIRUS UNCOATING AND ARLY PRMABILIZATION 2573 (Sterling Research Group, Rensselaer, N.Y.). Rhodanine (2-thioxo-4-thiaolidinone), guanidine hydrochloride, gliotoxin, and dactinomycin were purchased from Sigma Chemical Co., St. Louis, Mo. Alpha-sarcin, a toxic protein (molecular weight, 16,8) produced by Aspergillus giganteus, was given to us by D. M. Schuurmans (Department of Public Health, Lansing, Mich.), and hygromycin B (molecular weight, 527), produced by Streptomyces hygroscopicus, was obtained from Lilly Research Laboratories (Indianapolis, Ind.). Measurement of protein synthesis. Methionine-free medium (.2 ml per well) containing.2 mci of [35S]methionine (1,1 Ci/mmol) from the Radiochemical Centre (Amersham, ngland) was added to cells grown on 24-well dishes. The cell monolayer was incubated at 37 C for 1 h, and the medium was then removed. Cells were fixed with 5% trichloroacetic acid (TCA) and washed with ethanol. The fixed cell monolayer was dried and resuspended in.2 ml of.1 N NaOH-1% sodium dodecyl sulfate (SDS). The radioactivity was measured in a liquid scintillation counter (Intertecnique). Radiolabeling and purffication of poliovirus. HeLa cells were grown on 1-mm-diameter dishes. The cell monolayers were infected with poliovirus at a multiplicity of 5 PFU per cell in Dulbecco modified agle medium supplemented with 2% newborn calf serum. At 3 h postinfection, the cell monolayer was washed once with fresh methionine-free medium, and 3 ml of this medium containing 4 mci of [35S]methionine per dish was added. At 9 h postinfection, the cells were harvested and disrupted by freeing and thawing. The cellular extracts were centrifuged at 1, rpm for 3 min in an SS-34 Sorvall rotor at 4 C. The supernatants were layered onto 5 ml of a 15% sucrose cushion (5 mm NaCl, 1 mm Tris HCI [ph 7.4]) and centrifuged at 38, rpm for 2 h at 4 C in a T-865 Sorvall rotor. Pellets were resuspended in.5 ml of Dulbecco modified agle medium, and then Nonidet P-4 was added (1% final concentration). The virus solution was then centrifuged through a 15 to 45% linear sucrose gradient containing 5 mm NaCl and 1 mm Tris HCI (ph 7.4) at 25, rpm for 4 h in an AH-627 Sorvall rotor at 4 C. ach fraction contained 1.5 ml, of which 5,u was used for determining the peak of radioactive virus. Selected fractions were pooled and centrifuged at 38, rpm for 2 h in a T-865 rotor at 4 C, and the pellet was resuspended in.5 ml of Dulbecco modified agle medium. HeLa cells were infected with [35S]methionine-labeled poliovirus and incubated at 37 C. Unattached virus was removed by washing with phosphate-buffered saline (PBS), and the cell monolayer was treated with 5,ug of proteinase K per ml for 3 min at 4 C. Cells were collected, washed with PBS, and precipitated with 1% TCA. The radioactivity retained on GF/C filters was determined. Measurement of poliovirus attachment to HeLa cells. HeLa cells grown on 35-mm-diameter dishes were precooled on ice for 15 min and then were infected with [35S]methioninelabeled poliovirus and incubated at 4 C. At various times, the cell monolayers were washed twice with ice-cold PBS to remove unbound virus and the cells were collected (all these processes were done at 4 C). Cells were precipitated with 1% TCA, and the radioactivity retained on GF/C filters was measured. stimation of [3H]heparin uptake by HeLa cells. HeLa cell monolayers were incubated with [3H]heparin (.49 mci/mg; NN Research Products, Du Pont de Nemours, Dreieich, Germany) in 24-well plates. At 2 h postinfection, the cells were fixed with 5% TCA and then washed twice with a. c.c a on TIM (minutes) CL c t TIM (minutes) FIG. 1. (A) ffect of inhibitors on poliovirus attachment to HeLa cells. Cells were infected with [35S]methionine-labeled poliovirus in the absence of inhibitor (U) or in the presence of 2 plg of DPC per ml (O), 25 p.g of Ro 9-41 per ml (), or 1,±g of WIN per ml () and incubated at 4 C as described in Materials and Methods. HeLa cells were harvested, and the TCA-precipitable radioactivity retained on GF/C filters was counted at selected times postinfection. (B) ffect of inhibitors on poliovirus entry into cells. HeLa cells were infected with [35S]methionine-labeled poliovirus and incubated at 37 C in the absence or presence of the compounds described in panel A. Cells were collected and treated with proteinase K at the indicated times as described in Materials and Methods. The TCAprecipitable radioactivity retained on GF/C filters was measured. ethanol. Dried precipitated cells were resuspended in.2 ml of.1 N NaOH-1% SDS, and the radioactivity was estimated in an Intertecnique liquid scintillation counter. RSULTS Attachment and entry of poliovirus in the presence of uncoating inhibitors. We measured the binding of [35S]methionine-labeled poliovirus to HeLa cells at 4 C in the presence of WIN 51711, DPC, and Ro 9-41 to analye the effect of these agents on poliovirus attachment and entry. Poliovirus attached to cells at control levels in the presence of any of the three inhibitors tested (Fig. 1A). The internaliation of virus particles into HeLa cells at 37 C was not blocked by these inhibitors (Fig. 1B), indicating that these two early events are not the target of these compounds. This agrees with the findings that the uncoating process is the step blocked by these agents (15, 18, 24). ffect of WIN 51711, DPC, and Ro 9-41 on the simultaneous entry of alpha-sarcin and viral particles into cells. When virion particles enter cells, there is a parallel coentry of a number of enymes and protein toxins that are in the culture medium (4, 6, 13, 14, 28). Since protein synthesis is inhibited by these toxins, which have ribosomes as their target, this suggests that the toxins must be located in the cytoplasm (6, 14). The presence of alpha-sarcin together with poliovirus particles induced a profound and rapid blockade of protein synthesis (Fig. 2). Neither poliovirus particles nor alpha-sarcin alone were able to perform this inhibition. Strikingly, increasing concentrations of WIN 51711, DPC, or Ro 9-41 reversed this inhibition, suggesting that the toxin does not reach the cytoplasm when these uncoating inhibitors are present. This occurred even under high concentrations of alpha-sarcin (Fig. 3).
3 2574 ALMLA T AL. J. VIROL. CH3CH2S CI H3 NC )NQ cci a (CH2) )A -i ~ J I A n-%. I I W 4 81( 2 DPC (pg/ml) Ro-9 41 (pg/mi) WIN (pg/mi) FIG. 2. ffect of different concentrations of the uncoating inhibitors on the early permeabiliation. Cell monolayers were placed in methionine-free medium and mock infected or infected with poliovirus at a multiplicity of infection of 25 in presence or absence of 5,ug of alpha-sarcin per ml under different concentrations of the inhibitory compounds DPC, Ro 9-41, and WIN At 1 h postinfection, protein synthesis was measured as described in Materials and Methods. Symbols:, uninfected cells;, uninfected cells plus alpha-sarcin; A, poliovirus-infected cells; A, poliovirus-infected cells plus alpha-sarcin. We tested the effect of this phenomenon on other agents that block poliovirus growth by inhibiting poliovirus RNA synthesis, e.g., guanidine, gliotoxin, and Ro 9-179, to analye the selectivity of the inhibition of alpha-sarcin coentry with poliovirus. None of these compounds had any effect on the early permeabiliation of cells to alpha-sarcin (Table 1). The partial effect observed with Ro was mainly due to the inhibition observed with this compound in control cells. Rhodanine, a selective inhibitor of echovirus type 12 uncoating (12), and dactinomycin, an inhibitor of cellular but not viral RNA synthesis, were also devoid of effect (Table 1). A number of animal viruses including 1 DPC Ro-9 41 WIN J F- 5 -I I- C. -i o- 25 A - a ALPHA-SARCIN (Pg/ml) ALPHA-SARCIN (pg/mi) ALPHA-SARCIN (pg/mi) FIG. 3. ffect of uncoating inhibitors on the early membrane leakiness. HeLa cell monolayers were mock infected or infected with poliovirus at a multiplicity of infection of 25 PFU per cell in the absence or presence of 5,ug of DPC per ml, 1,ug of Ro 9-41 per ml, or 1,ug of WIN per ml. After 1 h of incubation at 37 C, protein synthesis was estimated as described in Materials and Methods. Percentage of control is expressed as [35S]methionine counts per minute in infected cells divided by the counts per minute in uninfected cells in the presence () or absence () of uncoating inhibitors.
4 VOL. 65, 1991 VIRUS UNCOATING AND ARLY PRMABILIZATION 2575 HeLa cells TABL 1. ffect of a number of compounds on the early permeabiliation induced by poliovirus infectiona Compound Control Amt of (35S]methionine incorporated (cpm) +Alpha-sarcin (% of control) Uninfected 19, ,78 (13) Poliovirus infected 84,7 22,183 (26) Poliovirus infected DPC (1,ug/ml) 52,894 57,774 (19) Poliovirus infected DPC (8,ug/ml) 6,246 62,951 (14) Poliovirus infected Ro 9-41 (15,ug/ml) 4,53 35,44 (87) Poliovirus infected Ro 9-41 (1,ug/ml) 52,233 5,971 (98) Poliovirus infected WIN (1,ug/ml) 86,128 78,797 (91) Poliovirus infected Ro (1,ug/ml) 5,622 26,979 (53) Poliovirus infected Rhodanine (2 jig/ml) 83,62 18,635 (22) Poliovirus infected Guanidine (3 mm) 87,212 19,44 (22) Poliovirus infected Gliotoxin (.15 p.g/ml) 87,689 21,287 (24) Poliovirus infected Dactinomycin (5,ug/ml) 66,26 12,4 (18) a HeLa cells grown in 24-well culture dishes were mock infected or infected with poliovirus type 1 at a multiplicity of infection of 25. The indicated compounds and 5 jlg of alpha-sarcin per ml were added as indicated to the methionine-free medium from the beginning of infection. At 1 h postinfection, proteins were labeled with [35Slmethionine for 1 h, and the radioactivity incorporated was measured. poliovirus permeabilie cells during the late phase of infection, the so-called "late membrane leakiness" (3). When inhibitors of poliovirus RNA replication were added early during infection but after poliovirus entry, they prevented the appearance of late membrane leakiness, measured by the entry of the nonpermeable inhibitor hygromycin B (Table 2). This effect did not occur when these agents were added 5 h postinfection. In fact, none of the antipicornavirus agents tested, including the uncoating inhibitors, were able to affect the late permeability to hygromycin B when they were added 5 h postinfection (Table 2). These results indicate that active viral replication is required to fully permeabilie cells late during infection and suggest that the blockade of alphasarcin entry by uncoating inhibitors during the early permeabiliation is very specific since these agents do not block late membrane permeability. [3H]heparin entry during early stages of poliovirus infection. The experiments described in the previous section showed that the macromolecule alpha-sarcin does not reach the cytoplasm in the presence of uncoating inhibitors. However, it is not known whether these macromolecules can still coenter with virion particles into endosomes and whether the exit of alpha-sarcin from endosomes is the actual step blocked by WIN 51711, DPC, and Ro We made use of radioactively labeled heparin to address this problem. Viruses not only promote the entry of proteins and other low-molecular-weight compounds into cells but also greatly enhanced the internaliation of polysaccharides (19). The presence of poliovirus enhanced the uptake of heparin into HeLa cells threefold (Fig. 4). This increased entry was not modified by the uncoating inhibitors that we used. All these findings fit well with the model depicted in Fig. 5, which suggests that viral particles are cointernalied with added compounds such as alpha-sarcin or heparin (4, 6, 13, 14). They would then pass to the cell interior through endosomes. The release of the viral genome into the cytoplasm (uncoating) or the exit of the cointernalied compounds would then be blocked by uncoating inhibitors. Thus, normal virus uncoating is required for the early permeabiliation by viruses. DISCUSSION The mechanism(s) used by naked viruses to cross cellular membranes and deliver their genomes in the cytoplasm or the nucleus remains obscure (1, 2, 4, 6). An attractive possibility is that virion particles have devices to permeabilie endosomal membranes (6). This possibility would ex- TABL 2. ffect of several compounds on the late permeabiliation induced by poliovirus infectiona Amt of [35S]methionine incorporated (cpm) when compound was added: HeLa cells Compound 1 h postinfection 5 h postinfection Control +Hygromycin B (% of control) Control +Hygromycin B (% of control) Uninfected 119, ,631 (97) 113, ,444 (113) Poliovirus infected 3,65 4,831 (16) 22,73 5,174 (23) Poliovirus infected DPC (1,ug/ml) 12,59 7,12 (59) 12,182 2,565 (21) Poliovirus infected Ro 9-41 (15 p.g/ml) 14,955 3,394 (23) 14,212 4,649 (33) Poliovirus infected Ro 9-41 (1,ug/ml) 17,775 6,39 (35) 25,44 4,935 (2) Poliovirus infected WIN (1,ug/ml) 25,769 5,6 (2) 22,2 7,218 (33) Poliovirus infected Rhodanine (2 jlg/ml) 4,315 7,59 (17) 23,731 4,245 (18) Poliovirus infected Ro (1,ug/ml) 7,81 41,283 (58) 18,63 2,781 (15) Poliovirus infected Guanidine (3 mm) 113,454 15,13 (93) 23,574 4,157 (18) Poliovirus infected Gliotoxin (.15,ug/ml) 51,368 32,84 (64) 23,372 3,76 (16) Poliovirus infected Dactinomycin (5 ±g/ml) 22,286 4,862 (22) a HeLa cells were grown in 24-well dishes and mock infected or infected with poliovirus type 1 at a multiplicity of infection of 25. Compounds were added to the medium at 1 or 5 h postinfection. In all cases, 1 mm hygromycin B was added at 5 h postinfection, and 3 min later, protein synthesis was measured as described in Materials and Methods.
5 2576 ALMLA T AL. J. VIROL. DPC Ro-9 41 WIN C c1 2 S C Il 4 21 CL la 1 D CL S1 A.s 3, 1. 2, I Hlheparin added (pci/ml) 3 O I I I Hlheparin added (pci/ml), 1, 2, I H]heparin added (pci/ml) FIG. 4. ffect of uncoating inhibitors on [3H]heparin entry. Monolayers of HeLa cells were mock infected or infected with poliovirus type 1 at a multiplicity of infection of 5 PFU per cell in the absence or presence of DPC (25,ug/ml), Ro 9-41 (1,ug/ml), or WIN (1,ug/ml). Several concentrations of [3H]heparin were added. The radioactivity internalied into HeLa cells was counted after 2 h of incubation at 37 C. Symbols: Uninfected cells with (O) or without (A) inhibitor; poliovirus-infected cells with () or without () inhibitor. plain why enveloped as well as naked viruses are able to permeabilie cell membranes to compounds present in the culture medium during early steps of virus infection (i.e., virus entry and uncoating) (4, 13). The entrance mechanism of these compounds into cells is probably their cointernaliation with virions (14). Most probably, endosomes are later permeabilied by virions in a process that remains obscure. Our present findings indicate that this early permeabiliation requires poliovirus uncoating. Thus, during uncoating, endosomes become permeabilied not only to the viral genome but also to other macromolecules entrapped inside them. Therefore, the uncoating process should be regarded as a series of well-defined events. After binding of poliovirus to its receptor, one or more virion proteins interact with the endosomal membrane. In fact, poliovirus protein VP4 is released in the membrane of the infected cells during the entry process and the amino terminus of VP1 is inserted into endosomal membranes (16). This mechanism may be similar to the interaction of the haptomer moiety of some protein toxins (21, 25), which promotes the entry of the effectomer moiety to the cytoplasm. In this regard, it is interesting to keep in mind that the haptomer molecule of these toxins can be replaced by virion particles (13), permitting effectors to pass to the cytoplasm in the presence of virions (13). Once the viral protein interacts with the endosomal membrane, it may become bound to it (9, 2). This event might trigger the Hepari n(hp) FIG. 5. Schematic representation of the mode of action of uncoating inhibitors. Alpha-sarcin or heparin when present in the culture medium is cointernalied with poliovirus particles in endosomes. Macromolecules are freed into the cytoplasm under normal conditions, but in the presence of uncoating inhibitors they are not liberated in the cytoplasm and follow the same fate as poliovirus particles.
6 VOL. 65, 1991 VIRUS UNCOATING AND ARLY PRMABILIZATION 2577 uncoating process by destabiliing virion particles. In turn, this may also increase membrane permeability (6). Thus, the uncoating step and the early membrane leakiness could be envisaged as coupled phenomena. When uncoating inhibitors are bound to virion particles filling the hydrophobic pocket (26, 28, 3, 33), they make some viral proteins more rigid, impeding the interaction of viral proteins with the endosomal membrane, a step needed for virus uncoating and membrane permeabiliation. The increased entry of polysaccharides combined with the coentry of protein toxins can be used as a convenient and easy test to identify uncoating inhibitors. Such compounds would not affect the increased entry of labeled polysaccharides, indicating that virus binding to the receptor and internaliation are not blocked. However, such compounds would impede the inhibition of translation due to the presence of alpha-sarcin or similar toxins. ACKNOWLDGMNTS We are indebted to M. G. Rossmann for the critical reading of this manuscript and to M. T. Kenny (Merrell Dow Research Institute), H. Ishitsuka and K. Yokose (Nippon Roche Research Center), and D. Pevear and M. A. McKinlay (Sterling Research Group) for the generous gifts of DPC, Ro 9-41, and WIN 51711, respectively. The technical assistance of M. Chorro is acknowledged. M..G. was the holder of an F.P.I. fellowship. Fundaci6n Ram6n Areces and CAICYT are acknowledged for financial support. RFRNCS 1. Acharya, R.,. Fry, D. Stuart, G. Fox, D. Rowlands, and F. Brown The three dimensional structure of foot-andmouth disease virus at 2.9 A resolution. Nature (London) 337: Badger, J., I. Minor, M. J. Kremer, M. A. Oliveira, T. J. Smith, J. P. Griffith, D. M. A. Guerin, S. Krishnaswamy, M. Luo, M. G. Rossmann, M. A. McKinlay, G. D. Diana, F. J. Dutko, M. Fancher, R. R. Rueckert, and B. A. Hein Structural analysis of a series of antiviral agents complexed with human rhinovirus 14. Proc. Natl. Acad. Sci. USA 85: Carrasco, L Membrane leakiness after viral infection and a new approach to the development of antiviral agents. Nature (London) 272: Carrasco, L Modification of membrane permeability induced by animal viruses early in infection. Virology 113: Carrasco, L., and M. steban Modification of membrane permeability in vaccinia virus-infected cells. Virology 117: Carrasco, L., M. J. Otero, and J. L. Castrillo Modification of membrane permeability by animal viruses. Pharm. Ther. 4: Coombs, K., and D. T. Brown The penetration of animal cells by viruses, p In L. Carrasco (ed.), Mechanisms of viral toxicity in animal cells. CRC Press, Inc., Boca Raton, Fla. 8. Croweli, R. L., and K. Lonberg-Holm (ed.) Virus attachment and entry into cells. American Society for Microbiology, Washington, D.C. 9. De Sena, J., and B. Mandel Studies on the in vitro uncoating of poliovirus. I. Characteriation of the modifying reaction. Virology 78: Diana, G. D., M. J. Otto, and M. A. McKinlay Inhibitors of picornavirus uncoating as antiviral agents. Pharm. Ther. 29: Dimmock, N. J Initial stages in infection with animal viruses. J. Gen. Virol. 59: ggers, H. J., M. A. Koch, A. Furst, G. D. Daves, J. J. Wilcynski, and K. Folkers Rhodanine: a selective inhibitor of the multiplication of echovirus 12. Science 167: Fernande-Puentes, C., and L. Carrasco Viral infection permeabilies mammalian cells to proteins. Cell 2: Fitgerald, D. J. P., R. Padmanabhan, I. Pastan, and M. C. Willingham Adenovirus-induced release of epidermal growth factor and Pseudomonas toxin into the cytosol of KB cells during receptor mediated endocytosis. Cell 32: Fox, M. P., M. J. Otto, and M. A. McKinlay Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug. Antimicrob. Agents Chemother. 3: Goldstein, J. L., R. G. W. Anderson, and M. S. Brown Coated pits, coated vesicles and receptor mediated endocytosis. Nature (London) 279: Fricks, C.., and J. M. Hogle Cell-induced conformational change in poliovirus: externaliation of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 64: Gonale, M.., M. J. Almela, M. Yacout, and L. Carrasco (3,4-Dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile inhibits poliovirus uncoating. Antimicrob. Agents Chemother. 34: Gonale, M.., and L. Carrasco Animal viruses promote the entry of polysaccharides with antiviral activity into cells. Biochem. Biophys. Res. Commun. 146: Guttman, N., and D. Baltimore A plasma membrane component able to bind and alter virions of poliovirus type 1: studies on cell-free alteration using a simplified assay. Virology 82: Hoch, D. H., M. RomeroMira, B.. hrlich, A. Finkelstein, B. R. DasGupta, and L. L. Simpson Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bylayers: relevance to translocation of proteins across membranes. Proc. Natl. Acad. Sci. USA 82: Hoekstra, D., and J. W. Kok ntry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes. Biosci. Rep. 9: Hogle, J. M., M. Chow, and D. J. Filman Three dimensional structure of poliovirus at 2.9 A resolution. Science 229: Ishitsuka, H., Y. T. Ninomiya, C. Ohsawa, M. Fujiu, and Y. Suhara Direct and specific inactivation of rhinovirus by chalcone Ro Antimicrob. Agents Chemother. 22: Kagan, B. L., A. Finkelstein, and M. Colombini Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes. Proc. Natl. Acad. Sci. USA 78: Luo, M., G. Vriend, G. Kamer, I. Minor,. Arnold, M. G. Rossmann, U. Boege, D. G. Scraba, G. M. Duke, and A. C. Palmenberg The atomic structure of Mengo virus at 3. A resolution. Science 235: Pevear, D. C., M. J. Fancher, P. J. Felock, M. G. Rossmann, M. S. Miller, G. Diana, A. M. Treasurywala, M. A. McKinlay, and F. J. Dutko Conformational change in the floor of the human rhinovirus canyon blocks adsorption to HeLa cell receptors. J. Virol. 63: Otero, M. J., and L. Carrasco Proteins are cointernalied with virion particles during early infection. Virology 16: Rossmann, M. G The structure of antiviral agents that inhibit uncoating when complexed with viral capsids. Antiviral Res. 11: Rossmann, M. G.,. Arnold, J. W. rickson,. A. Frankenberger, J. P. Griffith, H. J. Hecht, J.. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend Structure of a human common cold virus and functional relationship to other picornaviruses. Nature (London) 317: Rossmann, M. G., and J.. Johnson Icosahedral RNA virus structure. Annu. Rev. Biochem. 58: Seth, P., M. C. Willingham, and I. Pastan Adenovirusdependent increase in cell membrane permeability. J. Biol. Chem. 259: Sim,I. S., and K. G. McCullagh Potential targets for selective inhibition of viral replication, p In M. R. Hamden (ed.), Approaches to antiviral agents. McMillan, London. 34. Smith, T. J., M. J. Kremer, M. Luo, G. Vriend,. Arnold, G. Kamer, M. G. Rossmann, M. A. McKinlay, G. D. Diana, and M. J. Otto The site of attachment of human rhinovirus 14 for antiviral agents that inhibit uncoating. Science 233:
Virus Entry/Uncoating Delivery of genome to inside of a cell Genome must be available for first step of replication The Problem--barriers to infection Virion Barriers: Non-enveloped viruses capsid Enveloped
Cells and Their Environment Chapter 8 Cell Membrane Section 1 Homeostasis Key Idea: One way that a cell maintains homeostasis is by controlling the movement of substances across the cell membrane. Homeostasis
The DNA -> RNA -> Protein Pathway RNA Polymerase = enzyme that makes mrna from the DNA gene template http://www.bioteach.ubc.ca/molecularbiology/amonksflourishinggarden/ Plus-strand RNA Viruses Plus-strand
JOURNAL OF VIROLOGY, Apr. 1974, p. 881-887 Copyright i 1974 American Society for Microbiology Vol. 13, No. 4 Printed in U.SA. Effect of D-Penicillamine on Poliovirus Replication In HeLa Cells PARVIN MERRYMAN,
Proc. Nat. Acad. Sci. USA Vol. 68, No. 9, pp. 2057-2061, September 1971 Regulation of Protein Synthesis Initiation in HeLa Cells Deprived of Single ssential Amino Acids (valine/histidine/methiotiine/high-temperature
JOURNAL OF VIROLOGY, Sept. 1998, p. 7551 7556 Vol. 72, No. 9 0022-538X/98/$04.00 0 Copyright 1998, American Society for Microbiology. All Rights Reserved. NOTES The Poliovirus Empty Capsid Specifically
JOURNAL OF BACTERIOLOGY, Sept., 1965 Copyright 1965 American Society for Microbiology Vol. 90, No. 3 Printed in U.S.A. Mode of Action of the Antiviral Activity of Amantadine in Tissue Culture' C. E. HOFFMANN,
Proceedings of the National Academy of Science8 Vol. 66, No. 3, pp. 799-806, July 1970 Synthesis of Proteins in Cells Infected with Herpesvirus, VI. Characterization of the Proteins of the Viral Membrane*
Virology 269, 239 247 (2000) doi:10.1006/viro.2000.0258, available online at http://www.idealibrary.com on MINIREVIEW Cell Recognition and Entry by Rhino- and Enteroviruses Michael G. Rossmann,*,1 Jordi
APPLIED MICROBIOLOGY, Oct. 1969, p. 584-588 Copyright ( 1969 American Society for Microbiology Vol. 18, No. 4 Printed in U S A. Role of Interferon in the Propagation of MM Virus in L Cells DAVID J. GIRON
Chapt. 10 Cell Biology and Biochemistry Cell Chapt. 10 Cell Biology and Biochemistry The cell: Lipid bilayer membrane Student Learning Outcomes: Describe basic features of typical human cell Integral transport
JOURNAL OF VIROLOGY, Jan. 2005, p. 655 660 Vol. 79, No. 1 0022-538X/05/$08.00 0 doi:10.1128/jvi.79.1.655 660.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved. Interaction with
VIRUSES Many diseases of plants and animals are caused by bacteria or viruses that invade the body. Bacteria and viruses are NOT similar kinds of micro-organisms. Bacteria are classified as living organisms,
Chapter 4: Cell Membrane Structure and Function Plasma Membrane: Thin barrier separating inside of cell (cytoplasm) from outside environment Function: 1) Isolate cell s contents from outside environment
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 198, p. 381-385 99-224/8/8-381/5$2./ Vol. 4, No. 2 Inactivation of Poliovirus I (Brunhilde) Single Particles by Chlorine in Water D. G. SHARP* AND JENNY LEONG
The Cell Membrane Why cells must control materials Living cells must maintain homeostasis for survival. The cell membrane is the boundary between the cell and its environment. It is the cell membrane s
JOURNAL OF VIROLOGY, Nov. 1999, p. 9098 9109 Vol. 73, No. 11 0022-538X/99/$04.00 0 Copyright 1999, American Society for Microbiology. All Rights Reserved. Identification of Functional Domains in the 14-Kilodalton
Chapter 3b Cells Membrane transport - Student Notes 1 Transport are permeable Some molecules the membrane; others do 2 Types of Membrane Transport processes No cellular required Substance its processes
CHAPTER 8 MEMBRANE STRUCTURE AND FUNCTION Section B: Traffic Across Membranes 1. A membrane s molecular organization results in selective permeability 2. Passive transport is diffusion across a membrane
MEMBRANE STRUCTURE AND TRAFFIC Cell Membrane Structure and Function 4.1 How Is the Structure of a Membrane Related to Its Function? 4.1.1 The Plasma Membrane Isolates the Cell While Allowing Communication
INFECTION AND IMMUNITY, Dec. 197, p. 75-71 Copyright 197 American Society for Microbiology Vol., No. Printed in U.S.A. Accelerated Cytopathology in HeLa Cells Induced by Reovirus and Cycloheximide PHILIP
JOURNAL OF VIROLOGY, Aug. 1973, p. 265-274 Copyright 1973 American Society for Microbiology Vol. 12, No. 2 Printed in U.S.A. In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected
JOURNAL OF VIROLOGY, Apr. 2003, p. 4646 4657 Vol. 77, No. 8 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.8.4646 4657.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Ability of
Viruses Properties They are obligate intracellular parasites. Probably there are no cells in nature that escape infection by one or more kinds of viruses. (Viruses that infect bacteria are called bacteriophages.)
MOLECULAR MEDICINE REPORTS 1: 251-255, 2008 251 Effect of caffeine on the multiplication of DNA and RNA viruses MASAKI MURAYAMA 1, KAZUKO TSUJIMOTO 1,2, MISAO UOZAKI 1, YUKIKO KATSUYAMA 1, HISASHI YAMASAKI
Cellular Form and Function Concepts of cellular structure Cell surface Membrane transport Cytoplasm Modern Cell Theory All living organisms are composed of cells. the simplest structural and functional
6 Cell Membranes Valencia college 6 Cell Membranes Chapter objectives: The Structure of a Biological Membrane The Plasma Membrane Involved in Cell Adhesion and Recognition Passive Processes of Membrane
THE EFFECTS OF A TRYPTOPHAN-HISTIDINE DEFICIENCY IN A MUTANT OF ESCHERICHIA COLI MARGOT K. SANDS AND RICHARD B. ROBERTS Carnegie Institution of Washington, Department of Terrestrial Magnetism, Washington,
Cell Membrane Structure and Function What is the importance of having a cell membrane? I. Membrane Structure a. Membranes contain proteins, lipids, and carbohydrates (which are all types of macromolecules)
Chapter 13 Viruses, Viroids, and Prions Biology 1009 Microbiology Johnson-Summer 2003 Viruses Virology-study of viruses Characteristics: acellular obligate intracellular parasites no ribosomes or means
JOURNAL OF VIROLOGY, Aug. 1973, p. 386-396 Copyright 1973 American Society for Microbiology Vol. 12, No. 2 Printed in U.S.A. Penetration of Host Cell Membranes by Adenovirus 2 DENNIS T. BROWN' AND BYRON
Chapter 7: Membrane Structure and Function Concept 7.1 Cellular membranes are fluid mosaics of lipids and proteins 1. Phospholipids are amphipathic. Explain what this means. Name Period Amphipathic means
Onderstepoort J. vet. Res. (1968), 35 (1), 139-150 Printed in the Repub. of S. Afr. by The Government Printer, Pretoria ELECTRON MICROSCOPIC STUDY OF THE FORMATION OF BLUETONGUE VIRUS* G. LECATSAS, Veterinary
Membrane Structure and Function Chapter 7 Objectives Define the following terms: amphipathic molecules, aquaporins, diffusion Distinguish between the following pairs or sets of terms: peripheral and integral
MEMBRANE STRUCTURE AND FUNCTION (Please activate your clickers--question next slide) From Science, 3/12/2010: Pain s in the genes : Subtle changes to a certain gene seem to determine how sensitive people
Plasma Membrane Structure and Function The plasma membrane separates the internal environment of the cell from its surroundings. The plasma membrane is a phospholipid bilayer with embedded proteins. The
virology MCQs 1- A virus which causes AIDS is: a- Small pox virus. b- Coxsackie B virus. c- Mumps virus. d- Rubella virus. e- HIV-III virus. 2- A virus commonly transmitted by use of contaminated surgical
Cell Membrane Structure (1.3) IB Diploma Biology Essential idea: The structure of biological membranes makes them fluid and dynamic http://www.flickr.com/photos/edsweeney/6346198056/ 1.3.1 Phospholipids
2.4 Membranes 2.4.1 - Draw and label a diagram to show the structure of membranes Phospholipid Bilayer - This is arranged with the hydrophilic phosphate heads facing outwards, and the hydrophobic fatty
The Cell Cell theory (1838): 1. All organisms are composed of one or more cells, and the life processes of metabolism and heredity occur within these cells. 2. Cells are the smallest living things, the
IN: Unit 2: More on Matter & Energy in Ecosystems Macromolecules to Organelles to Cells Where are cells on the biological scale? Sub-Atomic Particles Atoms Molecules Macromolecules (proteins, lipids, nucleic
Cell Membrane Diagram Draw a diagram of the cell membrane. Please include (and label): - Phospholipid bilayer (hydrophilic and hydrophobic) Protein channel An ion pump Cholesterol Gylcoproteins* Define
Module 2C Membranes and Cell Transport All cells are surrounded by a plasma membrane. Eukaryotic cells also contain internal membranes and membrane- bound organelles. In this module, we will examine the
The Plasma Membrane - Gateway to the Cell 1 Photograph of a Cell Membrane 2 Cell Membrane The cell membrane is flexible and allows a unicellular organism to move 3 Homeostasis Balanced internal condition
The Plasma Membrane - Gateway to the Cell 1 Photograph of a Cell Membrane 2 Cell Membrane The cell membrane is flexible and allows a unicellular organism to move 3 Homeostasis Balanced internal condition
3- Cell Structure and Function How do things move in and out of cells? A Quick Review Taft College Human Physiology How do things move in and out of cells? Things may move through cell membranes by Passive
Basic Structure of a Cell 1 Introduction to Cells Cells are the basic units of organisms Cells can only be observed under microscope Basic types of cells: Animal Cell Plant Cell Bacterial Cell 2 Number
ISOLATION OF NONINFECTIOUS PARTICLES CONTAINING ROUS SARCOMA VIRUS RNA FROM THE MEDIUM OF ROUS SARCOMA VIRUS-TRANSFORMED NONPRODUCER CELLS* BY HARRIET LATHAM ROBINSONt VIRUS LABORATORY, UNIVERSITY OF CALIFORNIA,
OpenStax-CNX module: m44419 1 Bulk Transport * OpenStax This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 3.0 By the end of this section, you will be able
JOURNAL OF VIROLOGY, Aug. 2004, p. 8732 8745 Vol. 78, No. 16 0022-538X/04/$08.00 0 DOI: 10.1128/JVI.78.16.8732 8745.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Putative
The Annexin V Apoptosis Assay Development of the Annexin V Apoptosis Assay: 1990 Andree at al. found that a protein, Vascular Anticoagulant α, bound to phospholipid bilayers in a calcium dependent manner.
Chapter 7: Membrane Structure & Function 1. Membrane Structure 2. Transport Across Membranes 1. Membrane Structure Chapter Reading pp. 125-129 What are Biological Membranes? Hydrophilic head WATER They
Class: _ Date: _ Unit 1 Test: Cells Multiple Choice Identify the choice that best completes the statement or answers the question. 1) Which of the following is true of integral membrane proteins? A) They
J. gen. Virol. (1989), 70, 2799-2804. Printed in Great Britain 2799 Key words: rhinovirus, human type 14/5' non-coding region~in vitro translation Sequences in the 5" Non-coding Region of Human Rhinovims
Cellular Transport Notes About Cell Membranes All cells have a cell membrane Functions: a. Controls what enters and exits the cell to maintain an internal balance called homeostasis b. Provides protection
The Cell Membrane and Cellular Transportation Oct 20 7:07 PM Cell Membrane Forms a barrier between the cell and the external environment. Has three main functions: 1) helps the cell retain the molecules
Concept 7.1: Cellular membranes are fluid mosaics of lipids and proteins Lipids: Non-polar substances such as fat that contain C, H, O. Phospholipids: Lipid with phosphate group, very abundant in plasma
Human Umbilical Vein Endothelial Cell Manual INSTRUCTION MANUAL ZBM0079.03 SHIPPING CONDITIONS Human Umbilical Vein Endothelial Cells, cryopreserved Cryopreserved cells are shipped on dry ice and should
Coxsackievirus A9 VP1 Mutants with Enhanced or Hindered A Particle Formation and Decreased Infectivity Antero Airaksinen, Merja Roivainen and Tapani Hovi J. Virol. 2001, 75(2):952. DOI: 10.1128/JVI.75.2.952-960.2001.
ell biology 2017 version 13/1 2017 ote endosome vs lysosome handout Lecture 3: Text book Alberts et al.: hapter 12-14 (Topics covered by the lecture) A lot of reading! Focus on principles ell Biology interactive
ab139485 CytoPainter Golgi/ER Staining Kit Instructions for Use Designed to detect Golgi bodies and endoplasmic reticulum by microscopy This product is for research use only and is not intended for diagnostic
MEMBRANE STRUCTURE & FUNCTION Chapter 8 KEY CONCEPTS Cellular s are fluid mosaics of lipids and proteins Membrane structure results in selective permeability Passive transport is diffusion of a substance
The Role of the Cell Membrane in Transport diffusion: the spontaneous movement of particles from an area of higher concentration to an area of lower concentration Many people, young and old, enjoy a nice
Why would you want to study proteins associated with viruses or virus infection? Receptors Mechanism of uncoating How is gene expression carried out, exclusively by viral enzymes? Gene expression phases?
Mitochondrial DNA Isolation Kit Catalog Number KA0895 50 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials
BIOLOGY 12 - Cell Membrane and Cell Wall Function: Chapter Notes The cell membrane is the gateway into the cell, and must allow needed things such as nutrients into the cell without letting them escape.
Chapter 13B: Animal Viruses 1. Overview of Animal Viruses 2. DNA Viruses 3. RNA Viruses 4. Prions 1. Overview of Animal Viruses Life Cycle of Animal Viruses The basic life cycle stages of animal viruses
Chapter 3 Cells Introduction: A.The human body consists of 75 trillion cells that vary considerably in shape and size yet have much in common. B.Differences in cell shape make different functions possible.
AN INHIBITOR OF VIRAL ACTIVITY APPEARING IN INFECTED CELL CULTURES* BY MONTO Hot AND JOHN F. ENDERS RESEARCH DIVISION OF INFECTIOUS DISEASES, THE CHILDREN'S MEDICAL CENTER, AND THE DEPARTMENT OF BACTERIOLOGY
1. Peroxisomes are small, membrane-enclosed organelles that function in the degradation of fatty acids and in the degradation of H 2 O 2. Peroxisomes are not part of the secretory pathway and peroxisomal
BIOLOGY 12 - Cell Membrane and Cell Wall Function: Chapter Notes The cell membrane is the gateway into the cell, and must allow needed things such as nutrients into the cell without letting them escape.
Cell Membranes The cell / plasma membrane is. Selective in that it allows things in and some things out of the cell. Recall that phospholipids have hydrophobic and hydrophilic. The term to describe this
Secretion and Endocytosis Peter Takizawa Cell Biology Vesicular transport between organelles Glycosylation Protein sorting in the Golgi Endocytosis Secretory pathway delivers proteins and lipids to plasma
Plasma Membrane & Movement of Materials in Cells Why do cells need to control what enters and exits? Plasma membrane boundary between the cell and its environment Homeostasis maintaining the cells environment
Proceeding8 of the National Academy of Sciences Vol. 66, No. 2, pp. 572-576, June 1970 Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus, II. An RNA Polymerase in the Virion* David Baltimore, Alice
JOURNAL OF VIROLOGY, Aug. 1981, p. 348-358 Vol. 39, No. 2 0022-538X/81/080348-11$02.00/0 Specific Sindbis Virus-Coded Function for Minus-Strand RNA Synthesis DOROTHA L. SAWICKI,`* STANLY G. SAWICKI,' SIRKKA