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[34] Biosynthesis of myxovirus glycoproteins with special emphasis on mutants defective in glycoprotein processing
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[34] B i o s y n t h e s i s o f M y x o v i r u s G l y c o p r o t e i n s w i t h S p e c i a l E m p h a s i s o n M u t a n t s D e f e c t i v e in G l y c o p r o t e i n P r o c e s s i n g By H A N S - D I E T E R K L E N K
The surface glycoproteins of myxoviruses, i.e., the hemagglutinin and the neuraminidase of influenza viruses and the hemagglutinin-neuraminidase and the fusion protein of paramyxoviruses are integral membrane glycoproteins with their carbohydrate side chains attached to the polypeptide by N-glycosidic linkages. These glycoproteins play essential roles in the infection process by promoting adsorption of the virus to the host cell surface and penetration of the viral genome into the cytoplasm. Furthermore, the surface glycoproteins are the viral antigens that induce and react with neutralizing antibodies and are therefore the prime target of the immune response of the infected organism. Finally, the glycoproteins of myxoviruses are suitable models to study structure, function, and biosynthesis of membrane proteins in general. Synthesis of the myxovirus glycoproteins involves translation at membrane-bound ribosomes, insertion into the membrane of the rough endoplasmic reticulum, and transport to the plasma membrane from which the virus is released in a budding process. Insertion into the rough endoplasmic reticulum is fairly well understood with the influenza hemagglutinin, where it is mediated by a signal sequence at the amino terminus that is removed by cotranslational proteolytic cleavage. J Proteolytic cleavage at the posttranslational level has been observed with the influenza hemagglutinin, 2,3 the fusion protein, 4,5 and the hemagglutinin-neuraminidase of paramyxoviruses. 6 Proteases of trypsin specificity play an essential role in the cleavage reaction, and it depends on the presence of an appropriate enzyme, whether or not the glycoproteins are cleaved in a given cell. Posttranslational cleavage is necessary for the biological activities of the glycoproteins and thus for virus infectivity. It has also been found to be an important determinant for the spread of infection and for the pathogenicity of these viruses. 6,7 i j. McCauley, J. J. Skehel, K. Elder, M.-J. Gething, A. Smith, and M. Waterfield, in "Structure and Variation in Influenza Virus" (W. G. Laver and G. Air, eds.), p. 97. Elsevier/North-Holland, Amsterdam, 1980. 2 H.-D. Klenk, R. Rott, M. Orlich, and J. Bl6dorn, Virology 68, 426 (1975). 3 S. G. Lazarowitz and P. W. Choppin, Virology 68, 440 (1975). 4 M. Homma and M. Ohuchi, J. Virol. 12, 1457 (1973). 5 A. Scheid and P. W. Choppin, Virology 57, 475 (1974). 6 y . Nagai, H.-D. Klenk, and R. Rott, Virology 727 494 (1976). 7 F. X. Bosch, M. Orlich, H.-D. Klenk, and R. Rott, Virology 957 197 (1979).
METHODS IN ENZYMOLOGY, VOL. 96
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In addition to proteolytic cleavage, processing of the myxovirus glycoproteins involves also covalent attachment of fatty acids 8 and glycosylation. Glycosylation is initiated by the en bloc transfer of mannose-rich oligosaccharides from dolichol pyrophosphate to the asparagine residues of the polypeptides. This occurs in the rough endoplasmic reticulum. Upon arrival of the glycoproteins in the Golgi apparatus, some of the side chains undergo extensive modifications involving removal of mannose and attachment of galactose and fucose residues. Thus, two major types of carbohydrate side chains are found on the glycoproteins: the mannoserich type containing only mannose and glucosamine, and the complex type that contains in addition galactose and f u c o s e . 9
Viruses Most studies devoted to the biosynthesis of influenza virus glycoprot e i n s , 2,3,1°-15 have been carried out on two strains of influenza A viruses:
the Rostock strain of fowl plague virus [A/FPV/Rostock/34 (H7N 1)] 16 and strain A/WSN/33 (H1N1).17 These strains have been selected, because in tissue cultures they grow to relatively high titers of infectious virus [-109 plaque-forming units (PFU) per milliliter of tissue culture medium], form plaques, and suppress the synthesis of host cell proteins to allow the analysis of viral protein synthesis by metabolic labeling. Valuable information on the mechanism of proteolytic activation has been derived from the analysis of the primary structure of several hemagglutinins. These include again the hemagglutinins of fowl plague virus, 18 of the WSN strain, 19 of strain A/Chick/Germany/49 (H10N7), 2° and of a variety of hemagglutinins of the serotypes H2 and H3. 21 For the X-ray crystallos M. F. G. Schmidt, Virology 116, 327 (1982). 9 H.-D. Klenk and R. Rott, Curt. Top. Microbiol. Immunol. 90, 19 (1980). 10 S. G. Lazarowitz, R. W. Compans, and P. W. Choppin, Virology 46, 830 (1971). 11 H.-D. Klenk, R. Rott, and H. Becht, Virology 47, 579 (1972). 12 H.-D. Klenk, W. WOllert, R. Rott, and C. Scholtissek, Virology 57, 28 (1974). i3 H. J. Hay, Virology 60, 398 (1974). 14 R. W. Compans, Virology 51, 56 (1973). 15 K. Nakamura and R. W. Compans, Virology 93, 31 (1979). 16 W. SchRfer, Z. Naturforsch., B: Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 10B, 81 (1955). 17 T. Francis, Jr. and A. E. Moore, J. Exp. Med. 72, 717 (1940). 18 A. G. Porter, C. Barber, N. A. Carey, R. A. Hallewell, G. Threlfall, and J. S. Emtage, Nature (London) 282, 471 (1979). 19 A. C. Hiti, A. R. Davis, and D. P. Nayak, Virology 111, 113 (1981). 2o W. Garten, F. X. Bosch, D. Linder, R. Rott, and H.-D. Klenk, Virology 115, 361 (1981). 21 C. W. Ward, Curr. Top. Microbiol. Immunol. 94, 1 (1981).
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graphic analysis of the tertiary structure, also a H3 hemagglutinin has been used. 22 The biosynthesis of paramyxovirus glycoproteins has been analyzed primarily with Sendai virus 23 and Newcastle disease virus. 24-26 Seed stocks of myxoviruses are usually grown in the allantoic sac of 11-day-old embryonated chicken eggs that have been inoculated with about 104 PFU. Allantoic fluid is harvested 24-48 hr after infection, centrifuged at 2000 g for 15 min to remove debris, and stored at -80 °. Assays of Biological Activities of Viral Glycoproteins
Plaque Assay. 2 Virus infectivity is usually titrated by plaque assays in cultures of chick embryo cells, MDBK cells, or BHK cells (see below). Confluent monolayers on plastic petri dishes (5 cm in diameter) are rinsed with phosphate-buffered saline, pH 7.4, and infected with 0.2 ml of virus suspension diluted in the same buffer. After a 60-min adsorption at 37° the cultures are overlaid with 4 ml of medium 199 (Flow Laboratories) containing 0.08% bicarbonate and 0.7% Difco Bactoagar. Alternatively, reinforced Eagle's medium (REM) 27 with 0.7% Difco Bactoagar is used. When the viral glycoproteins are not activated by endogenous cellular proteases, trypsin is added to the overlay at final concentrations between 0.1 and 10/zg/ml. 2,3,5,6 Cultures are kept for 3 days at 37° and are then stained with 4 ml of phosphate-buffered saline (PBS), pH 7.4, containing 0.005% neutral red and 0.7% Difco Bactoagar. After 4 hr of incubation at 37°, plaques are counted. Hemagglutination Titration. Serial twofold dilutions of virus in a volume of 0.05 ml of PBS are prepared in plastic hemagglutination trays. A 0.05-ml amount of a 1% suspension of chicken red blood cells in PBS is added to each egg, and the HA titer is read after 45 min. With influenza viruses the assay is carried out at room temperature; with Sendal and Newcastle disease virus, at 4°. Hemadsorption. Cell cultures in plastic petri dishes are infected with virus. After the adsorption time, the inoculum is removed. The cultures are washed with warm PBS and incubated in culture medium. After an appropriate incubation period, the medium is removed and the cultures are washed with PBS. To each petri dish an equal amount of a suspension 22 I. A. Wilson, J. J. Skehel, and D. C. Wiley, Nature (London) 289, 366 (1981). 23 R. A. Lamb and P. W. Choppin, Virology 81, 371 (1977). 24 y . Nagai, H. Ogura, and H.-D. Klenk, Virology 69, 523 (1976). 25 y . Nagai and H.-D. Klenk, Virology 77, 125 (1977). 26 W. Garten, T. Kohama, and H.-D. Klenk, J. Gen. Virol. 51, 207 (1980). :7 R. H. Bablanian, H. J. Eggers, and I. Tamm, Virology 26, 100 (1965).
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of erythrocytes (0.5% in PBS) is added. After 10 min at room temperature, the suspension is removed and the cultures are thoroughly rinsed with PBS. The cell monolayers are then inspected under the microscope for adsorbed erythrocytes. Neuraminidase Assay. The enzyme is incubated for 30 min at 37° in 0.5 ml of buffer with fetuin at a final concentration of 100 /zg of Nacetylneuraminic acid. 2s Free neuraminic acid is measured by the thiobarbituric acid method, z9 One neuraminidase unit is defined as the amount of enzyme that releases 1 nmol of N-acetylneuraminic acid in 1 rain at 37°. Hemolysis Assay. The capacity of a myxovirus to induce membrane fusion can be measured by its hemolytic activity. With paramyxoviruses, the virus sample in 1 ml of PBS is mixed with 2 ml of a 2% suspension of chicken erythrocytes in PBS. After incubation for 60 min at 37°, the erythrocytes are removed by centrifugation, and the optical density of the supernatant is measured at 540 nm. 6 The fusion capacity of influenza viruses has an acidic pH optimum. With these viruses, the hemolysis assay is therefore carried out at pH 5.0-6.0 depending on the virus strain used. 3o Virus Replication in Cell Cultures
Cell Cultures. To analyze the individual steps in virus replication and assembly, myxoviruses are grown in cell cultures that are propagated as monolayers in plastic petri dishes or tissue culture flasks. Permanent cell lines frequently used for the propagation of influenza and paramyxoviruses are Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK) cells. 31 The BHK21 line of baby hamster kidney cells 31 is another permanent cell line that has been successfully used to study the biosynthesis of Sendal and Newcastle disease virus proteins. Such cultures are grown in REM containing 10% calf serum or, preferentially, fetal calf serum. Primary cultures of chicken embryo cells are also used for the growth of myxoviruses. Such cultures are prepared from 11day-old embryos after trypsinization and growth in tissue culture medium supplemented with calf serum. Confluent cell sheets form after 24-48 hr of incubation at 37° in an atmosphere of 5% CO2 in air. Infection of Cell Cultures. Monolayers in petri dishes are washed once with warm sterile PBS and are then exposed to the virus at a multiplicity 2s R. Drzeniek, J. T. Seto, and R. Rott, Biochim. Biophys. Acta 128, 547 (1966). D. Aminoff, Biochem. J. 81, 384 (1961). 3o R. T. C. Huang, R. Rott, and H.-D. Klenk, Virology 110, 243 (1981). 31 S. H. Madin and N. B. Darby, Jr., Proc. Soc. Exp. Biol. Med. 98, 574 (1958). 32 M. G. P. Stoker and I. A. McPherson, Virology 14, 359 (1961).
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of 10-50 PFU/ceI1. Adsorption is allowed to proceed for 40-60 min at 37 °. After adsorption the inoculum fluid is removed, fresh growth medium is added, and the dishes are incubated at the appropriate growth temperature. Radioactive Labeling of Infected Cells. Conditions for metabolic labeling of viral proteins are optimal when the rate of viral protein synthesis has reached a maximum and shutoff of host protein synthesis approximates a minimum. Depending on the virus and the host cell used, it takes 4-10 hr after inoculation to reach this stage. To label viral proteins with radioactive amino acids, the culture medium is removed, and the cells are washed three times with medium lacking the unlabeled amino acids to be substituted in radioactive form. The radioactive amino acids are then added in the same medium, and the cultures are incubated for an appropriate time. Radioactive amino acids are used at the following concentration: 3H-labeled amino acids (e.g., leucine, valine, tyrosin), 10/zCi/ml; ~4C-labeled protein hydrolyzate, 2/zCi/ml; [35S]methionine, 10/xCi/ml. If the radioactive pulse (5-20 min) is followed by a chase period (1-2 hr), the radioactive medium is removed and the cells are washed twice with complete medium and incubated in the same medium. The carbohydrate side chains of the viral glycoproteins can be labeled with radioactive sugars. Specific carbohydrate markers that are not converted, or only little, into other radioactively labeled compounds, are the following sugars: [6-3H]glucosamine, [2-3H]mannose, [1-3H]galactose, [l3H]fucose, and [1-14C]glucosamine. The sugars must be added in quite high concentrations (100 /~Ci/ml) to the culture medium, because the intracellular pools of their metabolic intermediates in the glycosylation reactions are high and cannot be lowered by simple washing procedures. For the same reason, labeling periods must be quite long (at least 2 hr) in certain host cells, particularly in primary chick embryo cells, and it is difficult to perform pulse-chase experiments. Because glucose and glucosamine have the same uptake mechanism, the incorporation of radioactive glucosamine is significantly increased when glucose in the medium is replaced by fructose (10 mM) as an energy source. 33 Radioactive Labeling of Released Virus. For metabolic labeling of the proteins incorporated into mature virions, the radioactive isotopes are usually added immediately after inoculation and left in the cell culture medium until virus harvest (about 24 hr postinfection). When radioactive amino acids are used for labeling, the respective unlabeled amino acids should be reduced in the serum-free medium to 10-30% of their normal concentrations. When radioactive glucosamine is used, glucose is again 33 C. Scholtissek, R. Rott, and H.-D. Klenk,
Virology 63, 191 (1975).
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replaced in the medium by fructose (10 raM). 3H-Labeled sugars and amino acids are added to the medium at a concentration of 2/~Ci/ml, 14Clabeled sugars and amino acids at 0.5 ~Ci/ml. To prepare fatty acidlabeled virus particles, REM with 2% fetal calf serum and 10-50/zCi/ml [9,10-3H]palmitic acid is used. 8 Localization of Viral Proteins in the Infected Cell. The compartmentalization of viral glycoproteins has been analyzed by a variety of different approaches, notably cell fractionation and immune electron microscopy. Preparation of Cytoplasmic Membranes. The procedure of Caliguiri and Tamm 34 has been used to isolate the cytoplasmic membrane of cells infected with influenza viruses 12,14 and paramyxoviruses. 2324 The cells from 20 petri dishes (10 cm in diameter) are washed with PBS and scraped in cold reticulocyte standard buffer (RSB) (0.01 M KC1, 0.0015 M MgC12, 0.01 M Tris-HCl, pH 7.4), held for 20 min in an ice bath, and disrupted with approximately 20 strokes of a tight-fitting Dounce homogenizer. Nuclei and cell debris are removed by centrifugation at 950 g for 10 min, and the supernatant is mixed with an equal volume of 60% sucrose in RSB. The resulting extract in 30% sucrose is included in a discontinuous gradient of 0, 25, 30, 40, 45, and 60% sucrose in RSB solutions and centrifuged at 24,000 rpm for 19 hr in a SW27 rotor. Generally six bands can be discriminated that are located (1) on the top of the gradient; (2) in the 25% sucrose layer; (3) in the interphases between the 25% and the 30% layers; (4) between the 30% and 40% layer; (5) between the 40% and 45% layers; and (6) in the 60% bottom layer. As revealed by electron microscopy and marker enzymes, fractions 2 and 3 contain smooth membranes derived from the Golgi apparatus and the smooth endoplasmic reticulum, fraction 5 contains rough membranes, and fraction 6 contains free ribosomes. With the exception of the first one, all bands are collected with a 10-ml syringe by puncturing from the side of the tube. After collection, each sample is diluted with RSB and pelleted at 100,000 g for 2 hr. The cell fractions can then be analyzed by polyacrylamide gel electrophoresis for radioactively labeled viral proteins and by the assays described above for the various biological activities of the virus. Isolation of Plasma Membranes. Plasma membranes can be isolated from a variety of cells by the fluorescein mercuric acetate (FMA) method. 35 The procedure developed for the infected B H K cells is the following. 36 Monolayers of 15-20 petri dishes (10 cm in diameter) are washed three times with cold PBS and then placed in 0.003 M EDTA. After 5 min at room temperature, the cells are pipetted off the plastic, 34 L. A. Caliguiri and I. Tamm, Virology 42, 100 (1970). 35 L. Warren, M. C. Glick, and M. K. Nass, J. Cell. Comp. Physiol. 68, 269 (1967). 36 H.-D. Klenk and P. W. Choppin, Virology 38, 255 (1969).
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pelleted, and washed once with PBS and twice with 0.16 M NaCI. The cells are suspended in 2 ml of 0.16 M NaC1 and 2 ml of water, and allowed to swell with agitation for 10 min at room temperature. Then 6 ml of a saturated solution of FMA in 0.02 M Tris buffer (pH 8.0) is added, the suspension is kept at room temperature for 15 min and in an ice bath for another 15 min. After 15-25 strokes of gentle homogenization in a Dounce homogenizer, an equal volume of 60% (w/w) sucrose is added, and the homogenate is layered over 10 ml of a 45% sucrose solution. After centrifugation at 350 g for 40 min, the 30% layer, which contains mainly wholecell ghosts or large pieces of membranes, is taken and pelleted by centrifugation at 2000 g for 2 hr. This membrane preparation is finally spun in a discontinuous sucrose gradient for 1 hr at 23,000 rpm in an SW27 rotor. Plasma membranes are found on the top and bottom of the 60% layer. Immune Electron Microscopy by the Indirect Ferritin-Antibody Labeling Technique. 37 Monolayer cultures in plastic petri dishes (3.5 cm in diameter) are infected with virus and incubated at 37°. Cells are sampled at appropriate times after infection and prefixed in situ with cold 0.25% glutaraldehyde in PBS for 15 min. After several PBS washings, the cell sheet is covered with 2% bovine serum albumin solution for 15 min. The excess albumin solution is aspirated, and after two PBS washings the cells are covered with 0.2 ml of the appropriate antibody preparation for 15 min at 25°. The excess antibody solution is washed off with PBS, and the cells are covered with 0.2 ml of the ferritin-antibody label for 15 min at 25 °. Untreated ferritin label is thoroughly washed with PBS. The cells are fixed with 2.5% glutaraldehyde in PBS for 1 hr, scraped, and pelleted by centrifugation at 1000 g for 10 min. The cell pellet is postfixed in 1% osmic acid, dehydrated in ethanol, and embedded in Epon 812. Mutants of Influenza Virus with Defects in Glycoprotein Transport Transport, glycosylation, and proteolytic cleavage are tightly coupled events in the biosynthesis of myxovirus glycoproteins. This has been demonstrated by the analysis of mutants of influenza virus that have temperature-sensitive defects in the transport of the hemagglutinin. 38,39 Chemical Mutagenesis of Influenza A Virus. The mutants with a defective hemagglutinin transport have been derived from the Rostock strain of fowl plague virus. 4°-42 The chemical mutagenesis by 5-fluoroura37 j. Lohmeyer, L. T. Talens, and H.-D. Klenk, J. Gen. Virol. 42, 73 (1979). 3s j. Lohmeyer and H.-D. Klenk, Virology 93, 134 (1979). 39 H.-D. Klenk, W. Garten, W. Keil, H. Niemann, F. X. Bosch, R. T. Schwarz, C. Scholtissek, and R. Rott, 1CN-UCLA Symp. Mol. Cell. Biol. 21, 193 (1981). 40 C. Scholtissek, R. Kruczinna, R. Rott, and H.-D. Klenk, Virology 58, 317 (1974).
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cil (5-FU) was performed by the following protocol originally described by Simpson and Hirst. 43 Chick embryo cell cultures infected with fowl plague virus at low multiplicity (-0.04) were incubated at 33° for 24 hr in virus growth medium containing 3 mM 5-FU, and virus released into the medium was harvested. Isolation o f ts Mutants. Treated or untreated fowl plague virus was appropriately diluted to give about 25 PFU per plate, and a sufficient number of monolayer cultures were inoculated to assure a minimum screening of at least 1000 plaques. Virus was adsorbed at room temperature for 30 min, after which agar overlay medium lacking neutral red was added. Infected cultures were incubated at 33° for 3-4 days before a second agar overlay containing neutral red was added. Distinct plaques were counted, and their borders were carefully traced on the plastic plate; the plates were then shifted to a 40° incubator for 18-24 hr. These plaques showing a minimal size increase after temperature shift were provisionally classified as foci containing ts virus and were selected for further testing. Plaque-derived virus was stored at 4° in 2-ml volumes of growth medium containing 20% normal chicken serum. To verify the ts character of provisional ts clones, 0.2 ml of undiluted plaque suspension was inoculated into each of two culture dishes (6 cm in diameter), which were subsequently overlaid with agar medium lacking neutral red. These cultures were incubated at 33° and 40°, respectively. Under these conditions most ts mutants produced confluent plaques in the low-temperature plating, whereas in the plate incubated at 40°, they formed no plaques or a few, usually small, foci. Two separate stocks of each ts mutant were prepared by passage of virus in chick embryo cell cultures for 2 days at 33° using as inoculum an aliquot of the original plaque suspension (temperature-shift screening) and virus derived from the plaques of the second 33° plating. These "master stocks" were assayed for PFU content and plating efficiency at 40° as described in the following sections. Preparation o f ts Mutant Stocks. The ts mutant was first recloned under agar overlay in chick embryo cell cultures incubated at 33°. Virus was picked from two or more well-isolated distinct plaques and suspended in 1 ml of a 2% albumin solution; 0.2 ml of this suspension was injected into embryonated eggs, which were incubated at 33° for 48 hr. Infectivity Assays and Determination of Plaquing Efficiency at 40 °. Fluids to be assayed for plaque-forming titer were diluted, and cell mono4t C. Scholtissek and A. C. Bowles, Virology 67, 576 (1975). 42 I. Koennecke, Thesis, Justus-Liebig-Universit/it, Giessen (1981). 43 R. W. Simpson and G. K. Hirst, Virology 35, 41 (1968).
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layers were inoculated with 1-ml volumes of diluted material. Inocula were adsorbed at room temperature for 30 rain, after which agar overlay medium lacking neutral red was added. The assay plates were incubated at 33° and 40 °, respectively. A second agar medium containing neutral red was added on day 3 or 4, and the plates were incubated for an additional 24 hr at the same temperatures previously employed. Distinct plaques were counted. All mutants chosen for study showed plaquing efficiencies at the restrictive temperature (40°) of 10-4 or less, as determined by dividing the 40 ° PFU titer by the 33° titer.
Protease Activation Mutants of Paramyxoviruses Mutants with alterations in glycoprotein processing have also been isolated with paramyxoviruses. The fusion protein of these mutants has an altered sensitivity to proteolytic activation. Cells that do not contain the appropriate enzyme to cleave the fusion protein, and thus do not allow multiple replication cycles of the virus, provide a suitable system for the isolation of such mutants. Isolation o f Sendai Virus Mutants Activated by Chymotrypsin and Elastase. 44 Unlike wild type, which can be activated by trypsin, the fusion protein of these mutants is cleaved by proteases of different specificities substituted to the cell culture medium. Sendai virus was mutagenized by treatment with nitrous acid. 4s Undiluted egg-grown virus, 0.5 ml, was incubated with 0.25 ml of 1 M sodium acetate, pH 4.4, and 0.25 ml of freshly prepared 4 M sodium nitrite. This treatment caused a decrease in virus infectivity, as determined by plaque assay with trypsin, of 1.5 logs in 1 min, 3.5 logs in 2 min, and 5.5 logs in 3 min. Virus treated for 1 min was used for mutant selection. Thirteen MDBK monolayers in 6-cm petri dishes received 0.5 ml of inocula containing ca. 1 x l04 PFU. After the adsorption period, the cells were incubated with REM containing chymotrypsin at a concentration of 1 ~g/ml to activate and permit multiple cycle growth of any mutant virus susceptible to this enzyme. After 5 days, 0.5 ml of the medium from each dish was used to inoculate a fresh MDBK monolayer. Within 3 days, two of these 13 cultures developed cytopathic effects typical of Sendal virus infection, and only these two cultures produced hemagglutinin. The harvests from these cultures were then tested for plaque formation with chymotrypsin, l ~g/ml, in the agar overlay. In contrast to wild-type virus, which does not form plaques under these conditions, these two inocula produced plaques. Individual plaques were 44 A. Scheid and P. W. Choppin, Virology 69, 265 (1976). 45 K. W. Mundry and A. Gierer, Z. Vererbungsl. 89, 614 (1958).
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selected and subjected to a second plaque purification, and stocks of virus were propagated in MDBK cells with chymotrypsin, 1 /~g/ml, added to the medium. Mutants activated by elastase were prepared by essentially the same procedure. Isolation of Mutants of Newcastle Disease Virus with Altered Protease Sensitivity of the Fusion Protein .46 The fusion protein of these mutants is cleaved by an endogenous protease of the host cell that is unable to activate wild-type virus. Mutants were induced in egg-grown virus (strains LaSota and Ulster) by treatment with 1 M sodium nitrite at pH 4.4 as described above. For mutant selection, monolayer cultures containing 10 6 MDBK cells were inoculated at a multiplicity of 10-3 and incubated with 5 ml of serum-free medium. After 3 days, 0.5 ml of the medium was used to inoculate a fresh culture, which was then incubated with serumfree medium for another 3 days. With both strains, in 1 out of about 500 twofold passages carried out in this way, virus production could be observed in the second passage as indicated by a distinct cytopathogenic effect and the release of hemagglutinin into the medium (128 HAU/ml). This virus was then subjected to three plaque passages in the same cell line. 46 W. Garten, W. Berk, Y. Nagai, R. Rott, and H.-D. Klenk, J. Gen. Virol. 50, 135 (1980).
[35] M e t h o d s for A s s a y o f C e l l u l a r R e c e p t o r s for P i c o r n a v i r u s e s By
RICHARD
L.
CROWELL,
DAVID
L.
KRAH, JOHN MAPOLES,
and
BURTON J. LANDAU
The picornaviruses are small (24-28 nm), single-stranded RNA-containing, nonenveloped viruses comprising over 170 immunologically distinct human viruses and a multitude of animal viruses. The several genera include the human enteroviruses (polioviruses, coxsackieviruses A and B, and echoviruses) and rhinoviruses, the murine cardioviruses, and the viruses of foot-and-mouth disease (FMDV).J Specific receptors for each of the several virus species exist on various cells in culture. 2-4 For example, the three poliovirus immunotypes compete for a receptor that is t R. 2 R. 3 K. 4 B.
R. Rueckert, Compr. Virol. 6, 131 (1976). L. Crowell, J. Bacteriol. 91, 198 (1966). L o n b e r g - H o l m , R. L. Crowell, and L. Philipson, Nature (London) 259, 679 (1976). Baxt and H. L. B a c h r a c h , Virology 104, 42 (1980).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
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[34] B i o s y n t h e s i s o f M y x o v i r u s G l y c o p r o t e i n s w i t h S p e c i a l E m p h a s i s o n M u t a n t s D e f e c t i v e in G l y c o p r o t e i n P r o c e s s i n g By H A N S - D I E T E R K L E N K
The surface glycoproteins of myxoviruses, i.e., the hemagglutinin and the neuraminidase of influenza viruses and the hemagglutinin-neuraminidase and the fusion protein of paramyxoviruses are integral membrane glycoproteins with their carbohydrate side chains attached to the polypeptide by N-glycosidic linkages. These glycoproteins play essential roles in the infection process by promoting adsorption of the virus to the host cell surface and penetration of the viral genome into the cytoplasm. Furthermore, the surface glycoproteins are the viral antigens that induce and react with neutralizing antibodies and are therefore the prime target of the immune response of the infected organism. Finally, the glycoproteins of myxoviruses are suitable models to study structure, function, and biosynthesis of membrane proteins in general. Synthesis of the myxovirus glycoproteins involves translation at membrane-bound ribosomes, insertion into the membrane of the rough endoplasmic reticulum, and transport to the plasma membrane from which the virus is released in a budding process. Insertion into the rough endoplasmic reticulum is fairly well understood with the influenza hemagglutinin, where it is mediated by a signal sequence at the amino terminus that is removed by cotranslational proteolytic cleavage. J Proteolytic cleavage at the posttranslational level has been observed with the influenza hemagglutinin, 2,3 the fusion protein, 4,5 and the hemagglutinin-neuraminidase of paramyxoviruses. 6 Proteases of trypsin specificity play an essential role in the cleavage reaction, and it depends on the presence of an appropriate enzyme, whether or not the glycoproteins are cleaved in a given cell. Posttranslational cleavage is necessary for the biological activities of the glycoproteins and thus for virus infectivity. It has also been found to be an important determinant for the spread of infection and for the pathogenicity of these viruses. 6,7 i j. McCauley, J. J. Skehel, K. Elder, M.-J. Gething, A. Smith, and M. Waterfield, in "Structure and Variation in Influenza Virus" (W. G. Laver and G. Air, eds.), p. 97. Elsevier/North-Holland, Amsterdam, 1980. 2 H.-D. Klenk, R. Rott, M. Orlich, and J. Bl6dorn, Virology 68, 426 (1975). 3 S. G. Lazarowitz and P. W. Choppin, Virology 68, 440 (1975). 4 M. Homma and M. Ohuchi, J. Virol. 12, 1457 (1973). 5 A. Scheid and P. W. Choppin, Virology 57, 475 (1974). 6 y . Nagai, H.-D. Klenk, and R. Rott, Virology 727 494 (1976). 7 F. X. Bosch, M. Orlich, H.-D. Klenk, and R. Rott, Virology 957 197 (1979).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
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In addition to proteolytic cleavage, processing of the myxovirus glycoproteins involves also covalent attachment of fatty acids 8 and glycosylation. Glycosylation is initiated by the en bloc transfer of mannose-rich oligosaccharides from dolichol pyrophosphate to the asparagine residues of the polypeptides. This occurs in the rough endoplasmic reticulum. Upon arrival of the glycoproteins in the Golgi apparatus, some of the side chains undergo extensive modifications involving removal of mannose and attachment of galactose and fucose residues. Thus, two major types of carbohydrate side chains are found on the glycoproteins: the mannoserich type containing only mannose and glucosamine, and the complex type that contains in addition galactose and f u c o s e . 9
Viruses Most studies devoted to the biosynthesis of influenza virus glycoprot e i n s , 2,3,1°-15 have been carried out on two strains of influenza A viruses:
the Rostock strain of fowl plague virus [A/FPV/Rostock/34 (H7N 1)] 16 and strain A/WSN/33 (H1N1).17 These strains have been selected, because in tissue cultures they grow to relatively high titers of infectious virus [-109 plaque-forming units (PFU) per milliliter of tissue culture medium], form plaques, and suppress the synthesis of host cell proteins to allow the analysis of viral protein synthesis by metabolic labeling. Valuable information on the mechanism of proteolytic activation has been derived from the analysis of the primary structure of several hemagglutinins. These include again the hemagglutinins of fowl plague virus, 18 of the WSN strain, 19 of strain A/Chick/Germany/49 (H10N7), 2° and of a variety of hemagglutinins of the serotypes H2 and H3. 21 For the X-ray crystallos M. F. G. Schmidt, Virology 116, 327 (1982). 9 H.-D. Klenk and R. Rott, Curt. Top. Microbiol. Immunol. 90, 19 (1980). 10 S. G. Lazarowitz, R. W. Compans, and P. W. Choppin, Virology 46, 830 (1971). 11 H.-D. Klenk, R. Rott, and H. Becht, Virology 47, 579 (1972). 12 H.-D. Klenk, W. WOllert, R. Rott, and C. Scholtissek, Virology 57, 28 (1974). i3 H. J. Hay, Virology 60, 398 (1974). 14 R. W. Compans, Virology 51, 56 (1973). 15 K. Nakamura and R. W. Compans, Virology 93, 31 (1979). 16 W. SchRfer, Z. Naturforsch., B: Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 10B, 81 (1955). 17 T. Francis, Jr. and A. E. Moore, J. Exp. Med. 72, 717 (1940). 18 A. G. Porter, C. Barber, N. A. Carey, R. A. Hallewell, G. Threlfall, and J. S. Emtage, Nature (London) 282, 471 (1979). 19 A. C. Hiti, A. R. Davis, and D. P. Nayak, Virology 111, 113 (1981). 2o W. Garten, F. X. Bosch, D. Linder, R. Rott, and H.-D. Klenk, Virology 115, 361 (1981). 21 C. W. Ward, Curr. Top. Microbiol. Immunol. 94, 1 (1981).
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graphic analysis of the tertiary structure, also a H3 hemagglutinin has been used. 22 The biosynthesis of paramyxovirus glycoproteins has been analyzed primarily with Sendai virus 23 and Newcastle disease virus. 24-26 Seed stocks of myxoviruses are usually grown in the allantoic sac of 11-day-old embryonated chicken eggs that have been inoculated with about 104 PFU. Allantoic fluid is harvested 24-48 hr after infection, centrifuged at 2000 g for 15 min to remove debris, and stored at -80 °. Assays of Biological Activities of Viral Glycoproteins
Plaque Assay. 2 Virus infectivity is usually titrated by plaque assays in cultures of chick embryo cells, MDBK cells, or BHK cells (see below). Confluent monolayers on plastic petri dishes (5 cm in diameter) are rinsed with phosphate-buffered saline, pH 7.4, and infected with 0.2 ml of virus suspension diluted in the same buffer. After a 60-min adsorption at 37° the cultures are overlaid with 4 ml of medium 199 (Flow Laboratories) containing 0.08% bicarbonate and 0.7% Difco Bactoagar. Alternatively, reinforced Eagle's medium (REM) 27 with 0.7% Difco Bactoagar is used. When the viral glycoproteins are not activated by endogenous cellular proteases, trypsin is added to the overlay at final concentrations between 0.1 and 10/zg/ml. 2,3,5,6 Cultures are kept for 3 days at 37° and are then stained with 4 ml of phosphate-buffered saline (PBS), pH 7.4, containing 0.005% neutral red and 0.7% Difco Bactoagar. After 4 hr of incubation at 37°, plaques are counted. Hemagglutination Titration. Serial twofold dilutions of virus in a volume of 0.05 ml of PBS are prepared in plastic hemagglutination trays. A 0.05-ml amount of a 1% suspension of chicken red blood cells in PBS is added to each egg, and the HA titer is read after 45 min. With influenza viruses the assay is carried out at room temperature; with Sendal and Newcastle disease virus, at 4°. Hemadsorption. Cell cultures in plastic petri dishes are infected with virus. After the adsorption time, the inoculum is removed. The cultures are washed with warm PBS and incubated in culture medium. After an appropriate incubation period, the medium is removed and the cultures are washed with PBS. To each petri dish an equal amount of a suspension 22 I. A. Wilson, J. J. Skehel, and D. C. Wiley, Nature (London) 289, 366 (1981). 23 R. A. Lamb and P. W. Choppin, Virology 81, 371 (1977). 24 y . Nagai, H. Ogura, and H.-D. Klenk, Virology 69, 523 (1976). 25 y . Nagai and H.-D. Klenk, Virology 77, 125 (1977). 26 W. Garten, T. Kohama, and H.-D. Klenk, J. Gen. Virol. 51, 207 (1980). :7 R. H. Bablanian, H. J. Eggers, and I. Tamm, Virology 26, 100 (1965).
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of erythrocytes (0.5% in PBS) is added. After 10 min at room temperature, the suspension is removed and the cultures are thoroughly rinsed with PBS. The cell monolayers are then inspected under the microscope for adsorbed erythrocytes. Neuraminidase Assay. The enzyme is incubated for 30 min at 37° in 0.5 ml of buffer with fetuin at a final concentration of 100 /zg of Nacetylneuraminic acid. 2s Free neuraminic acid is measured by the thiobarbituric acid method, z9 One neuraminidase unit is defined as the amount of enzyme that releases 1 nmol of N-acetylneuraminic acid in 1 rain at 37°. Hemolysis Assay. The capacity of a myxovirus to induce membrane fusion can be measured by its hemolytic activity. With paramyxoviruses, the virus sample in 1 ml of PBS is mixed with 2 ml of a 2% suspension of chicken erythrocytes in PBS. After incubation for 60 min at 37°, the erythrocytes are removed by centrifugation, and the optical density of the supernatant is measured at 540 nm. 6 The fusion capacity of influenza viruses has an acidic pH optimum. With these viruses, the hemolysis assay is therefore carried out at pH 5.0-6.0 depending on the virus strain used. 3o Virus Replication in Cell Cultures
Cell Cultures. To analyze the individual steps in virus replication and assembly, myxoviruses are grown in cell cultures that are propagated as monolayers in plastic petri dishes or tissue culture flasks. Permanent cell lines frequently used for the propagation of influenza and paramyxoviruses are Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK) cells. 31 The BHK21 line of baby hamster kidney cells 31 is another permanent cell line that has been successfully used to study the biosynthesis of Sendal and Newcastle disease virus proteins. Such cultures are grown in REM containing 10% calf serum or, preferentially, fetal calf serum. Primary cultures of chicken embryo cells are also used for the growth of myxoviruses. Such cultures are prepared from 11day-old embryos after trypsinization and growth in tissue culture medium supplemented with calf serum. Confluent cell sheets form after 24-48 hr of incubation at 37° in an atmosphere of 5% CO2 in air. Infection of Cell Cultures. Monolayers in petri dishes are washed once with warm sterile PBS and are then exposed to the virus at a multiplicity 2s R. Drzeniek, J. T. Seto, and R. Rott, Biochim. Biophys. Acta 128, 547 (1966). D. Aminoff, Biochem. J. 81, 384 (1961). 3o R. T. C. Huang, R. Rott, and H.-D. Klenk, Virology 110, 243 (1981). 31 S. H. Madin and N. B. Darby, Jr., Proc. Soc. Exp. Biol. Med. 98, 574 (1958). 32 M. G. P. Stoker and I. A. McPherson, Virology 14, 359 (1961).
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of 10-50 PFU/ceI1. Adsorption is allowed to proceed for 40-60 min at 37 °. After adsorption the inoculum fluid is removed, fresh growth medium is added, and the dishes are incubated at the appropriate growth temperature. Radioactive Labeling of Infected Cells. Conditions for metabolic labeling of viral proteins are optimal when the rate of viral protein synthesis has reached a maximum and shutoff of host protein synthesis approximates a minimum. Depending on the virus and the host cell used, it takes 4-10 hr after inoculation to reach this stage. To label viral proteins with radioactive amino acids, the culture medium is removed, and the cells are washed three times with medium lacking the unlabeled amino acids to be substituted in radioactive form. The radioactive amino acids are then added in the same medium, and the cultures are incubated for an appropriate time. Radioactive amino acids are used at the following concentration: 3H-labeled amino acids (e.g., leucine, valine, tyrosin), 10/zCi/ml; ~4C-labeled protein hydrolyzate, 2/zCi/ml; [35S]methionine, 10/xCi/ml. If the radioactive pulse (5-20 min) is followed by a chase period (1-2 hr), the radioactive medium is removed and the cells are washed twice with complete medium and incubated in the same medium. The carbohydrate side chains of the viral glycoproteins can be labeled with radioactive sugars. Specific carbohydrate markers that are not converted, or only little, into other radioactively labeled compounds, are the following sugars: [6-3H]glucosamine, [2-3H]mannose, [1-3H]galactose, [l3H]fucose, and [1-14C]glucosamine. The sugars must be added in quite high concentrations (100 /~Ci/ml) to the culture medium, because the intracellular pools of their metabolic intermediates in the glycosylation reactions are high and cannot be lowered by simple washing procedures. For the same reason, labeling periods must be quite long (at least 2 hr) in certain host cells, particularly in primary chick embryo cells, and it is difficult to perform pulse-chase experiments. Because glucose and glucosamine have the same uptake mechanism, the incorporation of radioactive glucosamine is significantly increased when glucose in the medium is replaced by fructose (10 mM) as an energy source. 33 Radioactive Labeling of Released Virus. For metabolic labeling of the proteins incorporated into mature virions, the radioactive isotopes are usually added immediately after inoculation and left in the cell culture medium until virus harvest (about 24 hr postinfection). When radioactive amino acids are used for labeling, the respective unlabeled amino acids should be reduced in the serum-free medium to 10-30% of their normal concentrations. When radioactive glucosamine is used, glucose is again 33 C. Scholtissek, R. Rott, and H.-D. Klenk,
Virology 63, 191 (1975).
[34]
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replaced in the medium by fructose (10 raM). 3H-Labeled sugars and amino acids are added to the medium at a concentration of 2/~Ci/ml, 14Clabeled sugars and amino acids at 0.5 ~Ci/ml. To prepare fatty acidlabeled virus particles, REM with 2% fetal calf serum and 10-50/zCi/ml [9,10-3H]palmitic acid is used. 8 Localization of Viral Proteins in the Infected Cell. The compartmentalization of viral glycoproteins has been analyzed by a variety of different approaches, notably cell fractionation and immune electron microscopy. Preparation of Cytoplasmic Membranes. The procedure of Caliguiri and Tamm 34 has been used to isolate the cytoplasmic membrane of cells infected with influenza viruses 12,14 and paramyxoviruses. 2324 The cells from 20 petri dishes (10 cm in diameter) are washed with PBS and scraped in cold reticulocyte standard buffer (RSB) (0.01 M KC1, 0.0015 M MgC12, 0.01 M Tris-HCl, pH 7.4), held for 20 min in an ice bath, and disrupted with approximately 20 strokes of a tight-fitting Dounce homogenizer. Nuclei and cell debris are removed by centrifugation at 950 g for 10 min, and the supernatant is mixed with an equal volume of 60% sucrose in RSB. The resulting extract in 30% sucrose is included in a discontinuous gradient of 0, 25, 30, 40, 45, and 60% sucrose in RSB solutions and centrifuged at 24,000 rpm for 19 hr in a SW27 rotor. Generally six bands can be discriminated that are located (1) on the top of the gradient; (2) in the 25% sucrose layer; (3) in the interphases between the 25% and the 30% layers; (4) between the 30% and 40% layer; (5) between the 40% and 45% layers; and (6) in the 60% bottom layer. As revealed by electron microscopy and marker enzymes, fractions 2 and 3 contain smooth membranes derived from the Golgi apparatus and the smooth endoplasmic reticulum, fraction 5 contains rough membranes, and fraction 6 contains free ribosomes. With the exception of the first one, all bands are collected with a 10-ml syringe by puncturing from the side of the tube. After collection, each sample is diluted with RSB and pelleted at 100,000 g for 2 hr. The cell fractions can then be analyzed by polyacrylamide gel electrophoresis for radioactively labeled viral proteins and by the assays described above for the various biological activities of the virus. Isolation of Plasma Membranes. Plasma membranes can be isolated from a variety of cells by the fluorescein mercuric acetate (FMA) method. 35 The procedure developed for the infected B H K cells is the following. 36 Monolayers of 15-20 petri dishes (10 cm in diameter) are washed three times with cold PBS and then placed in 0.003 M EDTA. After 5 min at room temperature, the cells are pipetted off the plastic, 34 L. A. Caliguiri and I. Tamm, Virology 42, 100 (1970). 35 L. Warren, M. C. Glick, and M. K. Nass, J. Cell. Comp. Physiol. 68, 269 (1967). 36 H.-D. Klenk and P. W. Choppin, Virology 38, 255 (1969).
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pelleted, and washed once with PBS and twice with 0.16 M NaCI. The cells are suspended in 2 ml of 0.16 M NaC1 and 2 ml of water, and allowed to swell with agitation for 10 min at room temperature. Then 6 ml of a saturated solution of FMA in 0.02 M Tris buffer (pH 8.0) is added, the suspension is kept at room temperature for 15 min and in an ice bath for another 15 min. After 15-25 strokes of gentle homogenization in a Dounce homogenizer, an equal volume of 60% (w/w) sucrose is added, and the homogenate is layered over 10 ml of a 45% sucrose solution. After centrifugation at 350 g for 40 min, the 30% layer, which contains mainly wholecell ghosts or large pieces of membranes, is taken and pelleted by centrifugation at 2000 g for 2 hr. This membrane preparation is finally spun in a discontinuous sucrose gradient for 1 hr at 23,000 rpm in an SW27 rotor. Plasma membranes are found on the top and bottom of the 60% layer. Immune Electron Microscopy by the Indirect Ferritin-Antibody Labeling Technique. 37 Monolayer cultures in plastic petri dishes (3.5 cm in diameter) are infected with virus and incubated at 37°. Cells are sampled at appropriate times after infection and prefixed in situ with cold 0.25% glutaraldehyde in PBS for 15 min. After several PBS washings, the cell sheet is covered with 2% bovine serum albumin solution for 15 min. The excess albumin solution is aspirated, and after two PBS washings the cells are covered with 0.2 ml of the appropriate antibody preparation for 15 min at 25°. The excess antibody solution is washed off with PBS, and the cells are covered with 0.2 ml of the ferritin-antibody label for 15 min at 25 °. Untreated ferritin label is thoroughly washed with PBS. The cells are fixed with 2.5% glutaraldehyde in PBS for 1 hr, scraped, and pelleted by centrifugation at 1000 g for 10 min. The cell pellet is postfixed in 1% osmic acid, dehydrated in ethanol, and embedded in Epon 812. Mutants of Influenza Virus with Defects in Glycoprotein Transport Transport, glycosylation, and proteolytic cleavage are tightly coupled events in the biosynthesis of myxovirus glycoproteins. This has been demonstrated by the analysis of mutants of influenza virus that have temperature-sensitive defects in the transport of the hemagglutinin. 38,39 Chemical Mutagenesis of Influenza A Virus. The mutants with a defective hemagglutinin transport have been derived from the Rostock strain of fowl plague virus. 4°-42 The chemical mutagenesis by 5-fluoroura37 j. Lohmeyer, L. T. Talens, and H.-D. Klenk, J. Gen. Virol. 42, 73 (1979). 3s j. Lohmeyer and H.-D. Klenk, Virology 93, 134 (1979). 39 H.-D. Klenk, W. Garten, W. Keil, H. Niemann, F. X. Bosch, R. T. Schwarz, C. Scholtissek, and R. Rott, 1CN-UCLA Symp. Mol. Cell. Biol. 21, 193 (1981). 40 C. Scholtissek, R. Kruczinna, R. Rott, and H.-D. Klenk, Virology 58, 317 (1974).
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cil (5-FU) was performed by the following protocol originally described by Simpson and Hirst. 43 Chick embryo cell cultures infected with fowl plague virus at low multiplicity (-0.04) were incubated at 33° for 24 hr in virus growth medium containing 3 mM 5-FU, and virus released into the medium was harvested. Isolation o f ts Mutants. Treated or untreated fowl plague virus was appropriately diluted to give about 25 PFU per plate, and a sufficient number of monolayer cultures were inoculated to assure a minimum screening of at least 1000 plaques. Virus was adsorbed at room temperature for 30 min, after which agar overlay medium lacking neutral red was added. Infected cultures were incubated at 33° for 3-4 days before a second agar overlay containing neutral red was added. Distinct plaques were counted, and their borders were carefully traced on the plastic plate; the plates were then shifted to a 40° incubator for 18-24 hr. These plaques showing a minimal size increase after temperature shift were provisionally classified as foci containing ts virus and were selected for further testing. Plaque-derived virus was stored at 4° in 2-ml volumes of growth medium containing 20% normal chicken serum. To verify the ts character of provisional ts clones, 0.2 ml of undiluted plaque suspension was inoculated into each of two culture dishes (6 cm in diameter), which were subsequently overlaid with agar medium lacking neutral red. These cultures were incubated at 33° and 40°, respectively. Under these conditions most ts mutants produced confluent plaques in the low-temperature plating, whereas in the plate incubated at 40°, they formed no plaques or a few, usually small, foci. Two separate stocks of each ts mutant were prepared by passage of virus in chick embryo cell cultures for 2 days at 33° using as inoculum an aliquot of the original plaque suspension (temperature-shift screening) and virus derived from the plaques of the second 33° plating. These "master stocks" were assayed for PFU content and plating efficiency at 40° as described in the following sections. Preparation o f ts Mutant Stocks. The ts mutant was first recloned under agar overlay in chick embryo cell cultures incubated at 33°. Virus was picked from two or more well-isolated distinct plaques and suspended in 1 ml of a 2% albumin solution; 0.2 ml of this suspension was injected into embryonated eggs, which were incubated at 33° for 48 hr. Infectivity Assays and Determination of Plaquing Efficiency at 40 °. Fluids to be assayed for plaque-forming titer were diluted, and cell mono4t C. Scholtissek and A. C. Bowles, Virology 67, 576 (1975). 42 I. Koennecke, Thesis, Justus-Liebig-Universit/it, Giessen (1981). 43 R. W. Simpson and G. K. Hirst, Virology 35, 41 (1968).
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layers were inoculated with 1-ml volumes of diluted material. Inocula were adsorbed at room temperature for 30 rain, after which agar overlay medium lacking neutral red was added. The assay plates were incubated at 33° and 40 °, respectively. A second agar medium containing neutral red was added on day 3 or 4, and the plates were incubated for an additional 24 hr at the same temperatures previously employed. Distinct plaques were counted. All mutants chosen for study showed plaquing efficiencies at the restrictive temperature (40°) of 10-4 or less, as determined by dividing the 40 ° PFU titer by the 33° titer.
Protease Activation Mutants of Paramyxoviruses Mutants with alterations in glycoprotein processing have also been isolated with paramyxoviruses. The fusion protein of these mutants has an altered sensitivity to proteolytic activation. Cells that do not contain the appropriate enzyme to cleave the fusion protein, and thus do not allow multiple replication cycles of the virus, provide a suitable system for the isolation of such mutants. Isolation o f Sendai Virus Mutants Activated by Chymotrypsin and Elastase. 44 Unlike wild type, which can be activated by trypsin, the fusion protein of these mutants is cleaved by proteases of different specificities substituted to the cell culture medium. Sendai virus was mutagenized by treatment with nitrous acid. 4s Undiluted egg-grown virus, 0.5 ml, was incubated with 0.25 ml of 1 M sodium acetate, pH 4.4, and 0.25 ml of freshly prepared 4 M sodium nitrite. This treatment caused a decrease in virus infectivity, as determined by plaque assay with trypsin, of 1.5 logs in 1 min, 3.5 logs in 2 min, and 5.5 logs in 3 min. Virus treated for 1 min was used for mutant selection. Thirteen MDBK monolayers in 6-cm petri dishes received 0.5 ml of inocula containing ca. 1 x l04 PFU. After the adsorption period, the cells were incubated with REM containing chymotrypsin at a concentration of 1 ~g/ml to activate and permit multiple cycle growth of any mutant virus susceptible to this enzyme. After 5 days, 0.5 ml of the medium from each dish was used to inoculate a fresh MDBK monolayer. Within 3 days, two of these 13 cultures developed cytopathic effects typical of Sendal virus infection, and only these two cultures produced hemagglutinin. The harvests from these cultures were then tested for plaque formation with chymotrypsin, l ~g/ml, in the agar overlay. In contrast to wild-type virus, which does not form plaques under these conditions, these two inocula produced plaques. Individual plaques were 44 A. Scheid and P. W. Choppin, Virology 69, 265 (1976). 45 K. W. Mundry and A. Gierer, Z. Vererbungsl. 89, 614 (1958).
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selected and subjected to a second plaque purification, and stocks of virus were propagated in MDBK cells with chymotrypsin, 1 /~g/ml, added to the medium. Mutants activated by elastase were prepared by essentially the same procedure. Isolation of Mutants of Newcastle Disease Virus with Altered Protease Sensitivity of the Fusion Protein .46 The fusion protein of these mutants is cleaved by an endogenous protease of the host cell that is unable to activate wild-type virus. Mutants were induced in egg-grown virus (strains LaSota and Ulster) by treatment with 1 M sodium nitrite at pH 4.4 as described above. For mutant selection, monolayer cultures containing 10 6 MDBK cells were inoculated at a multiplicity of 10-3 and incubated with 5 ml of serum-free medium. After 3 days, 0.5 ml of the medium was used to inoculate a fresh culture, which was then incubated with serumfree medium for another 3 days. With both strains, in 1 out of about 500 twofold passages carried out in this way, virus production could be observed in the second passage as indicated by a distinct cytopathogenic effect and the release of hemagglutinin into the medium (128 HAU/ml). This virus was then subjected to three plaque passages in the same cell line. 46 W. Garten, W. Berk, Y. Nagai, R. Rott, and H.-D. Klenk, J. Gen. Virol. 50, 135 (1980).
[35] M e t h o d s for A s s a y o f C e l l u l a r R e c e p t o r s for P i c o r n a v i r u s e s By
RICHARD
L.
CROWELL,
DAVID
L.
KRAH, JOHN MAPOLES,
and
BURTON J. LANDAU
The picornaviruses are small (24-28 nm), single-stranded RNA-containing, nonenveloped viruses comprising over 170 immunologically distinct human viruses and a multitude of animal viruses. The several genera include the human enteroviruses (polioviruses, coxsackieviruses A and B, and echoviruses) and rhinoviruses, the murine cardioviruses, and the viruses of foot-and-mouth disease (FMDV).J Specific receptors for each of the several virus species exist on various cells in culture. 2-4 For example, the three poliovirus immunotypes compete for a receptor that is t R. 2 R. 3 K. 4 B.
R. Rueckert, Compr. Virol. 6, 131 (1976). L. Crowell, J. Bacteriol. 91, 198 (1966). L o n b e r g - H o l m , R. L. Crowell, and L. Philipson, Nature (London) 259, 679 (1976). Baxt and H. L. B a c h r a c h , Virology 104, 42 (1980).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5