Journal of Virological Methods. 15 (1987) 273-277 Elsevier

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JVM OOS66

Homogeneous distribution of parainfluenza virus glyco~rot~ins demonstrated by immunogold-

labelling and light staining with uranyl acetate in electron microscopy

Francisco Hernandez’,Patricia Rivera and Yasuhiro Hosaka Deparmzenr of Preventive Medicine, Research Insrime for Microhiolo~iral Diseases, Osaka

University. Osaka, Japan

(Accepted 31 October 1986)

Parainfluenza virus; Sendai virus; Glycoprotein; Immunoelectron microscopy; Colloidal gold - _..-..

Two glycoproteins of parainfluenza 1 type, Sendai virus, namely haemagglu- tinin-neuraminidase (HN) and fusion (F) proteins, are needed for entry of the vi- rus into ceils and the induction of cell fusion (Choppin and Cornpans. 1975; Ho- saka et al., 1974; Ishida and Homma, 1978; Ozawa et al., 1979; @veil and Grandien. 1982). Thus, information on the distributions of I-IN and F glycopro- teins on virions is important for understanding the interactions of Sendai virus with cell membranes. Recently, the distribution of haemagglutinin (HA) and neura- minidase (NA) glycoproteins of influenza virus was determined by electron mi- croscopy by immunogold-1abeIling; NA gIycoproteins were detected in clusters, whereas HA appeared to be- uniformly distributed (Murti and Webster, 1986). However, we had difficulty in visualizing ail the immunogold particles bound to virions on negative or positive staining of preparations with 1% uranyl acetate (UA) by the usual procedure. We also found that nucleocapsids were frequently re- leased from native Sendai virions without fixation, but also spikeless areas on the surfaces of native virions were often seen. We developed, therefore. suitable con-

Correspondence to: F. Hernandez, Department of Preventive Medicine, Research Institute for Micro- biological Diseases, Osaka University. Suita, Osaka 565. Japan.

’ Recipient of a JICA Scholarship from the Unit of Electron Microscopy, University of Costa Rica. San Jose, Costa Rica. 2 Recipient of a Taniguchi Scholarship from the National Children’s Hospital of Costa Rica. San Jose, Costa Rica.

0166-0934/871$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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ditions for immuno-labelling and examined the distribution of HN and F glyco- proteins of Sendai virus.

Sendai virus was inoculated into the chorioallantoic cavity of IO-day-old chick embryos, and after 3 days incubation, the infected chorioallantoic fluid was cen- trifuged at a low speed. The resulting supernatant was centrifuged at 40,000 x g

for 30 min, and the pellet was suspended in phosphate-buffered saline (PBS), lay- ered on a discontinuous gradient of 30 and 50% sucrose, and centrifuged at 50,000 x s for 2.5 h. The band of virus above the 50%’ sucrose cushion was collected and recentrifuged in the same way. Finally, the sucrose was removed at 40,000 x g for 30 min. and the virus was resuspended in PBS.

Immuno-labelling was achieved by a modification of the method by Murti and Webster (1986). The purified virions (100.000 HAUlml) were diluted appropri- ately with Tris-buffered saline (TBS; 25 mM Tris buffer. pH 7.4. and 0.5 M NaCl). and fixed by addition of a half volume of a mixture of 3f% paraformaldehyde and 1% glutaraldehyde (Karnofsky, 1965) on a carbon-coated formvar film. This film had been treated with an ion cleaner (Eiko Engineering. Japan) to make it hy- drophilic. The excess fluid was then removed and the film was washed with TBS. It was then floated successively on a drop of 3% gelatin in TBS at 25°C for 1 h. a drop of 30-fold diluted monospecitic anti-F or anti-HN rabbit antiserum (Fukami et al.. 19X0) at 25°C for 1 h, and a drop of 25-fold diluted immunogold antiserum

FIN. I. Immunogold-lahelling of unlixed Sendal wrions with anti-F serum. Part of the virions was free

of immunogold particles (arrows) and a tail-like structure was seen (arrowhead) that appeared to bc

derived l’rom the area without gold particles (arrow) of the ume particle. Bar: 100 nm.

21s

Fig. 2. Immunogold-labelling with anti-HN serum of fixed Sendai virions. Positively (A) and nega- tively (B) stained images are shown. For details. see text. Bar: 100 nm.

(anti-rabbit IgGigoat IgG (EM GAB GS); Janssen, Belgium) with washings with TBS after each treatment. Finally, it was washed thoroughly with TBS. stained with 0.2% UA in deionized water for 5 min, and examined in a Hitachi HU12A elec- tron microscope. The conditions used in this procedure were judged to be optimal from the results of preliminary experiments.

Briefly, the results of these preliminary experiments were as follows. The use of

276

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Fig. 3. Immunogold-lahelling with anti-HN (A). anti-F (B), and normal (C) Sera of fixed Sendai vi-

rions. Light staining with 0.2% UA. Bar: 100 nm.

277

carbon-coated formvar films reduced the background number of gold particles than the case of uncoated films. Treatment with 3% gelatin gave a lower background number of gold particles than treatment with 0.1 or 1.0% gelatin. A temperature of 25°C during gelatin treatment was better than at 4”C, while at 37°C many nu- cleocapsids were released from unfixed virions. Fixation prevented release of nu- cleocapsids and also prevented possible capping of viral glycoproteins with anti- bodies and alteration of the envelope, changes that were frequently seen in unfixed virions (Fig. 1). The fixation procedure did not seem to affect the efficiency of im- muno-labelling. Positive staining of virions with 1 or 2% UA in ethanol solution sometimes obscured the location of gold particles, due to its dense staining of vi- rions, particularly their central areas, as seen in Fig. 2A. Negative staining with UA in aqueous solution obscured gold particles in peripheral areas surrounding virions (Fig. 2B). Light staining with 0.2% UA in aqueous solution allowed clear detection of almost all the gold particles bound to virions (Fig. 3A, B), in most paticles stained positively and some negative staining images. Thus, we used light staining.

Fig. 3A and B show Sendai virions treated first with anti-HN and anti-F anti- sera, respectively, and then with immunogold antibody. In both preparations, la- belled gold particles were homogeneously distributed on Sendai virions. Thus, these glycoproteins are both homogeneously distributed over the surface of the virions, and no particular structural or functional polarity of the envelopes of Sendai virus for their attachment to, and penetration into the host cells can be expected. Neg- ligible gold particles were observed on virions treated with normal rabbit serum (Fig. 3C).

Thus. the present procedure of immunoelectron microscopy will be generally useful for study of distributions of glycoproteins or proteins on the surface of vi- rions.

References

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Fukami, Y., Hosaka, Y. and Yamamoto, K. (1980) FEBS Let. 114. 342. Hosaka, Y., Semba. T. and Fukai, K. (1974) J. Gen. Viral. 25, 391.

Ishida. N. and Homma. M. (1978) Ad. Virus Res. 23. 349. Karnofsky. M.J. (1965) J. Cell. Biol. 27, 137a. Murti. K.G. and Webster, R.G. (1986) Virology 149. 36.

Orvell. C. and Grandien. M. (1982) J. Immunol. 129, 2779.

Ozawa. M.. Asano, A. and Okada. Y. (1979) Virology 99, 197.