Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein

Abstract

Infection of susceptible murine cells with the coronavirus mouse hepatitis virus type 4 (MHV4) results in extensive cell-cell fusion at pHs from 5.5 to 8.5. The endosomotropic weak bases chloroquine and ammonium chloride do not prevent MHV4 infection. In marked contrast, we have selected variants from a neural cell line persistently infected with MHV4 which are entirely dependent on acid pH to fuse host cells and are strongly inhibited by endosomotropic weak bases. Wild-type and variant viruses were compared at the level of the fusion-active surface (S) glycoprotein gene. Cloning and sequencing of each 4,131-base open reading frame predicted a total of eight amino acid differences which fell into three distinct clusters. Each S glycoprotein, when expressed from cDNA, was synthesized in equivalent amounts, and similar proportions were transported to the cell surface. Wild-type S induced cell-cell fusion at neutral pH, whereas variant S required prolonged exposure to acidic pH to induce fusion. Expression of hybrid S genes prepared by exchange of restriction fragments between wild-type and variant cDNAs revealed that elimination of neutral pH fusion was solely dependent on amino acid alterations at positions 1067 (Q to H), 1094 (Q to H), and 1114 (L to R). These changes lie within a predicted heptad repeat region of the transmembrane cleavage fragment of S (S2). These findings demonstrate that the pH dependence of coronavirus fusion is highly variable and that this variability can be determined by as few as three amino acid residues.

A conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions

“…The locus of the mutation that brings about the multiple changes in the S protein of Albl8 is distinct from that of any previously mapped MHV S mutation (Gallagher et al, 1991;Wang et al, 1992;Gombold et al, 1993;Fu and Baric, 1994). Serine 287 fails a little more than a third of the distance from the mature amino terminus of the S1 half of the molecule (Fig.…”

Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century

“…Potential mechanisms of action of CQ against coronaviruses are through underglycosylation of ACE2, which has been identified as a functional cellular receptor of SARS-CoV spike protein [33,117]. Alteration by CQ of the SARS-CoV spike protein is controversial [33,118]. A pH-related reduction of the transduction of SARS-CoV pseudotype viruses has also been suggested [33,119,120].…”

Molecular Interactions in the Assembly of Coronaviruses

“…The ectodomain of the S2 subunit, which is involved in the fusion process, contains two heptad repeat (HR) regions (de Groot et al, 1987a,b), a sequence motif characteristic of coiled coils. Mutations in the first (i.e., membrane-distal) HR region of the MHV S protein resulted in fusion-negative phenotypes (Luo and Weiss, 1998) or in a low-pH dependence for fusion (Gallagher et al, 1991), whereas mutations in the second HR region caused defects in S protein oligomerization and fusion ability (Luo et al, 1999). A fusion peptide has not yet been identified in any of the coronavirus spike proteins, but is predicted to be located at (Bosch et al, 2004b;Chambers et al, 1990) or within (Luo and Weiss, 1998) the amino terminus of the first HR region.…”