Cell Surface Antigen Induced by Influenza Virus

Nicholas Hahon, Herbert L. Eckert
1972 Infection and Immunity  
By immunofluorescence staining, a specific surface antigen induced by influenza virus was detected on clone 1-5C-4 cells. The antigen was neither related to hemagglutinin nor dependent on the production of infectious virus. Formation of the virus-specific cell surface antigen was dependent on protein synthesis but independent of viral ribonucleic acid replication. Virus-specific surface or membrane antigens have been induced by a variety of oncogenic and nononcogenic deoxyribonucleic acid (DNA)
more » ... onucleic acid (DNA) and ribonucleic acid (RNA) viruses on established cell cultures or on tumor cells (15, 19, 25, 26, 29, 39, 42) . The ability to induce early surface antigens on cells may be a property common to a wide variety of animal viruses. The mode of synthesis of virus-specific cell surface antigens and their relationship to viral components have not been fully clarified. Studies with a limited number of virus cell systems indicate, however, that the formation of these early antigens is dependent on protein synthesis but independent of either viral RNA or DNA replication (15, 19, 27, 28, 39, 40) . In view of evidence implicating mammalian cell membranes as sites of virus replication and of metabolic functions associated with these membranes (4, 10-12, 33, 34), the role of host cell membranes in virus replicative processes assumes added significance. Moreover, studies on the detection and characterization of cell surface antigens induced by viruses have helped enlarge our understanding of ensuing alterations in cell behavior (2, 7), determinants of cell immunological specificity (29) , and viral carcinogenesis (16). This report describes the induction of cell surface antigen by influenza virus and the sequence of its formation relative to other viral antigens. MATERIALS AND METHODS Cell cultures. Clone 1-5C-4 cells derived from a variant line of Chang's human conjunctival cell (41) were propagated with Eagle minimum essential medium (MEM) containing 10% fetal calf serum (FCS) and maintained with MEM and 5% FCS. For experimental purposes, cells were cultivated on 15-mm diameter cover slips inserted in flat-bottomed glass vials (19 by 65 mm). Approximately 106 cells, introduced into each vial, formed a complete cell monolayer within 24 hr of incubation at 35 C. Virus. The principal influenza virus strain employed in this study was Ao/PR8 obtained from the American Type Culture Collection, Rockville, Md. Stock virus preparation consisting of infectious allantoic fluids contained 1 X 108 cell-infecting units (CIU) of virus per ml when assayed by the immunofluorescent cell-counting technique. Infection of cells. Generally, virus diluted in phosphate-buffered saline (PBS), pH 7.2, was introduced onto confluent cell monolayers in 0.2-ml volumes at a multiplicity of infection (MOI) of 0.01. Attachment of inoculum onto cells was augmented by centrifugal force, 16,000 X g, for 12 min. Residual inoculum was removed, the cell monolayers were rinsed with PBS, and then 1 ml of maintenance medium was added. To demonstrate influenza cell surface antigen, infected cell monolayers were incubated at 35 C for 8 to 12 hr, rinsed with PBS, and, without fixation, stained by the direct immunofluorescence technique. Cell monolayers were fixed with acetone to demonstrate intracellular antigens. Preliminary tests showed that influenza cell surface antigen was unstainable within 5 min after acetone fixation. Immunofluorescence procedures. Virus antiserum was prepared in rabbits with initial intramuscular injections (2.0 ml) of an equal quantity of virus suspension and Freund complete adjuvant. At weekly intervals thereafter, animals were twice injected intravenously with 1.5 ml of virus suspension; they were exsanguinated 10 days after the last injection. The globulin fraction of antiserum, precipitated with ammonium sulfate, was conjugated with fluorescein isothiocyanate (0.02 mg/mg of protein), and then passed through a Sephadex G-25 column to remove unreacted dye. To reduce nonspecific fluorescence, 5 ml of conjugated globulin was diluted with an equal volume of PBS, adsorbed twice with 200 mg of acetone-dried mouse liver powder, and adsorbed once with 100 mg of chicken embryo powder. The general procedure for staining infected cells has been described previously (14) . Cover slip cell monolayers were examined with a Zeiss fluorescence microscope equipped with an FITC exciter and no. 50 and 65 barrier filters. With this optical system at a magnification of X400, the number of microscope fields contained in the area of a 15-mm diameter cover slip was 1,759. For each cover slip cell monolayer, 50 microscope fields were examined for fluorescent cells. 730 on May 8, 2020 by guest http://iai.asm.org/ Downloaded from
doi:10.1128/iai.6.5.730-735.1972 fatcat:ebw6gxagezh6phlu4ibppdeqku