We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold-or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that (i) the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and (ii) the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

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Essex C04 354, UKThe ability of Candida albicans to aggregate and adhere to biological surfaces is a topic of major biological and medical importance. One factor which has been implicated in such properties is the hydrophobic nature of the cell surface. Two simple spectroscopic techniques are described which permit the rapid determination of this property. The first involves the use of arylnaphthalenesulfonate, the fluorescence emission maximum of which was shown to be a sensitive indicator of dielectric polarity. This was used to identify the hydrophobic characteristics of the cell surface of C. albicans. The second technique involves the use of 90" RayleigkDebye light scattering as an indicator of the aggregation state of a fungal suspension. These techniques were then used to compare the surface properties of three different strains of C. albicans and the effects of culture conditions: the hydrophobicity of the strains varied, and galactose-based culture media promoted the greatest degree of cell surface hydrophobicity.

Chimeric (5) as is found in influenza virus-infected cells (2). To elucidate further some of the structural features involved in biogenesis, transport, and sorting of influenza HA, we have constructed and expressed chimeric cDNA clones in which the DNA encoding either the NH2 terminus or the COOH terminus of HA has been replaced with that of VSV G. In this report, we describe the properties of these chimeric proteins that have been expressed in CV1 cells and discuss the implication of these observations for translocation, sorting, and transport of the HA glycoprotein. MATERIALS AND METHODSVirus Strains, Cells, and Plasmid Vectors. CV1P and CV1 cells were grown in Dulbecco's modified Eagle's medium (DME medium) supplemented with 10% fetal bovine serum. Virus stocks of A/WSN/33 (HlN1) strain of influenza virus were prepared in Madin-Darby bovine kidney (MDBK) cells as described (6). SVHA2 [simian virus 40 (SV40) late-replacement vector expressing WSN HA] and SVSal-32 (SV40 defective in T-antigen expression) were grown in CV1 cells (7). pG1 and pGR125 containing VSV G cDNA were obtained from John K. Rose (8, 9).We expressed the chimeric HAG and GHA cDNA employing the late SV40 promoter as described for HA (10). To produce a lytic infection, SVSal32, which provides late gene function, was used as a helper and virus stock was prepared as described (7).Antibodies and Immunofluorescent Staining. Anti-HA monoclonal antibody (H15A13-18) against PR8 virus was obtained from W. Gerhard. Anti-WSN antibody was prepared from rabbits. Procedures for intracellular and surface staining using indirect immunofluorescence have been described (7).Radiolabeling of Infected Cells and Analysis of Polypeptides. At 40-48 hr after infection, cells were labeled for 5-6 hr by using 3 ml of methionine-free DME medium supplemented with 2% dialyzed fetal bovine serum and 50 ,Ci of L-[35S]methionine per ml (1 Ci = 37 gBq). For tunicamycin treatment, cells were first pretreated for 1 hr at 37°C with tunicamycin (2 ,ug/ml) in DME medium containing 2% fetal bovine serum and then labeled with L-[35S]methionine (50 ,Ci/ml) in DME medium containing tunicamycin (2 ,ug/ml).Subsequently, cells were washed twice with cold Tris (25 mM)-buffered saline, scraped, pelleted, and lysed in RIPA buffer (0.05 M Tris, pH 7.4/0.15 M NaCl/1% Triton X-100/1% sodium deoxycholate/0.1% NaDodSO4) (11, 12) for 10 min at 0°C. Nuclei were removed by centrifuging 15 min in an Eppendorf centrifuge and the supernatant was incubated for 1 hr at 4°C with 2 ,l of anti-WSN antibody. The antigen-antibody complexes were isolated by using protein ASepharose, washed four times, and analyzed on a 10% NaDodSO4/polyacrylamide gel (12-14).Endoglycosidase H (endo H) Treatment. At 40-48 hr after infection, cells were labeled for 2 hr with L-[35S]methionine as described above. The cells were then washed with methionine-free DME medium and incubated further in DME medium containing 2% fetal bovine serum for 3 hr. Cells were subsequently lysed and immunoprecipitated as described abo...

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