Arch Virol (1992) 122:307-316 _Amhives vi rology Springer-Verlag 1992 Printed in Austria Coronavirus IBV-induced membrane fusion occurs at near-neutral pH D. Li* and D. Cavanagh Agricultural and Food Research Council, Insitute for Animal Health, Division of Molecular Biology, Houghton Laboratory, Huntingdon, Cambridgeshire, U.K. Accepted June 21, 1991 Summary. The lysosomotropic agent NH4C1 caused a reduction of 80-95% in the number of chick kidney (CK) cells and Vero cells infected by infectious bronchitis virus (IBV) strain Beaudette, as determined by immunofluorescence at the end of the first replication cycle. Inhibition only occurred when NH4C1 was present during the first 2 h after infection. Syncytium formation was studied during replication of IBV-Beaudette in Vero cells. Some cell-ceU fusion occurred at pH 7.0 and pH 6.5 but it was optimal at pH 6.7. IBV strain UK/123/82 did not replicate in Vero cells and was studied in CK cells in which it grew well but without forming syncytia. In contrast to IBV-Beaudette, NH4C1 had vir- tually no effect on the replication of UK/123/82. The results show that the IBV spike glycoprotein induces membrane fusion at near neutral pH although some IBV strains may require a mildly acidic environment for the efficient uncoating of the virion RNA. Introduction Members of the monogeneric family Coronaviridae are enveloped, each species containing a large spike glycoprotein (S) which forms the bulbous surface projections, responsible for induction of membrane fusion 4, 20, 23, and a small membrane glycoprotein (M), most of which is not exposed at the outer virus surface. In IBV and some other coronaviruses the S protein is cleaved to form an amino-terminal S 1 and a carboxy-terminal $2 subunit 1, 22. A subset of the coronaviruses, which includes bovine coronavirus (BCV) and murine hepatitis virus (MHV) but not avian IBV, contains a third glycoprotein (HE) which has haemagglutinating and acetyl-esterase activities 22. The ribonu- * Present address: Harbin Veterinary Research Institute, Harbin, Heilongziang Province, Peoples Republic of China. 308 D. Li and D. Cavanagh cleoprotein (RNP) comprises a single, positive-sense RNA molecule of some 30,000 nucleotides surrounded by a phosphorylated nucleocapsid protein (N) 22. The study of membrane fusion induced by coronaviruses has been limited largely to MHV and BCV. Host cells infected with either virus fused to form syncytia when the culture medium was at pH 7.5 or greater but not at pH 7.0 or less 6, 8, 21. These results indicated that the membrane fusing activity of the spike glycoprotein (S) did not require triggering in an acidic environment, in contrast to orthomyxoviruses, togaviruses and flaviviruses 8, 9, 10, 12. Moreover, the results suggested that release of the RNP might occur following fusion of the virion envelope with the cell surface (plasmalemma) which can occur with paramyxoviruses 12, 16 and many mammalian retroviruses 14. Indeed, fusion of BCV with the plasmalemma has been observed 19. Fusion of coronaviruses with cell membranes has also been studied indirectly using lysosomotropic agents, for example NH4CI and chloroquine. These agents raise the pH of endosomes and lysosomes, in which viruses are located following endocytosis, and would be expected to inhibit the release of the RNP from an acid pH-requiring virus but not of a virus that was able to fuse at neutral pH. In agreement with the observations that BCV induced syncytia above pH 7.0 and fused with the plasmalemma, NH4C1 had little effect on the replication of BCV 18, 19. However, NH4C1 and chloroquine did inhibit 11 or delay 15-1 the replication of MHV3 and MHV-A59, respectively. To investigate further the conditions of coronavirus-induced membrane fu- sion we have examined the effect of acidic pH and NH4CI on the formation of syncytia and the early stages of infection, respectively, with IBV. Materials and methods Viruses and cells IBV-Beaudette, adapted for growth in Vero cells, was as previously described 1. Strain UK/123/82 had been passaged several times in chicken embryo tracheal organ cultures, and five times in embryos and primary chick kidney (CK) cells. Veto cells (ATCC No. CCL 81; Flow Laboratories, Irvine, Scotland) and CK cells were grown in M199 (Flow) and Eagles minimal essential medium (MEM; Gibco, Paisley, Scotland) respectively, sup- plemented with 10% foetal calf serum, 0.22% sodium bicarbonate and penicillin and streptomycin, in 5% COz. IBV-Beaudette was fitrated by plaque assay in Vero or CK cells. Cells were washed with MEM buffered with 20raM N-hydroxyethylpiperazine-N-2- ethanesulfonic acid (HEPES) and 0.08% sodium bicarbonate. Infected cells were overlaid with MEM containing 10% tryptose phosphate broth (Difco) in place of serum, 1% agar (Difco) and sodium bicarbonate at 0.22% and 0.15% for CK and Vero cells respectively. After 3 days at 37 C a second overlay was added which contained 0.003% neutral red.