The Avian Coronavirus Infectious Bronchitis Virus Undergoes Direct Low-pH-Dependent Fusion Activation during Entry into Host Cells

Abstract

Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronaviruscell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20°C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.For all enveloped viruses, a critical event during entry into cells is the fusion of the viral envelope with the membrane of the host cell (13). Our current understanding of viral fusion has been driven by fundamental problems first solved with influenza hemagglutinin (HA) (50). Whereas the trigger for HA-mediated fusion is the low pH of the endosome, other viruses (e.g., paramyxoviruses and most retroviruses) undergo a receptor-primed fusion with the plasma membrane at neutral pH (13).Coronaviruses (CoV) have recently received much attention due to the outbreak of severe acute respiratory syndrome (SARS) (22, 28), but there is little consensus as to whether coronavirus entry and fusion occur following endocytosis or at the plasma membrane (6,16,21,43). Coronaviruses are enveloped positive-strand RNA viruses that replicate in the cytoplasm (28). They have a distinctive set of club-shaped spikes on their envelope, and the spike protein (S) is the primary determinant of cell tropism and pathogenesis, being responsible (and apparently sufficient) for receptor binding and fusion (16). However, other envelope proteins are present: the M protein, the E protein, and (in some coronaviruses) an HE protein (28). The coronavirus S protein is categorized as a class I fusion protein, based on the presence of characteristic heptad repeats (3, 9, 26); as such, it shows features of the fusion proteins of influenza virus (HA), retroviruses (Env), and paramyxoviruses (F and HN), for which there is extensive characterization at the structural and biophysical levels (11).Al...

Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH

“…However, neuraminidase treatment of HMPV F-and G-expressing cells did not alter the requirement for low pH to induce fusion (data not shown), though it should be noted that the optimal pH for neuraminidase activity (approximately pH 5) was not employed, as this would make the analysis of pH effects on fusion difficult. It is also possible that strains of HMPV which form syncytia have F proteins which can be activated in the secretory pathway under certain circumstances, as was suggested for infectious bronchitis virus (12), since the pH of some secretory vesicles (49) is at the level found to trigger HMPV F-promoted membrane fusion in this study.…”

Characterization of the complete genome, antigenicity, pathogenicity, tissue tropism, and shedding of a recombinant avian infectious bronchitis virus with a ck/CH/LJL/140901-like backbone and an S2 fragment from a 4/91-like virus

“…membrane fusion unit of the ectodomain contains two heptad repeat regions, which interact to form the coiled-coil structure of the stalk (de Groot et al, 1987;Wickramasinghe et al, 2011), and a putative fusion peptide. After endocytosis, conformational changes in the S protein are triggered by exposure to acidic pH in the endosomes (Chu et al, 2006), thereby resulting in fusion of the viral envelope with the cellular membrane. The S2 domain is not involved mainly with binding to the host cell receptor, but the interplay between S1 and S2 might synergistically determine the avidity and specificity of viral attachment (de Hann et al, 2006;Promkuntod et al, 2013).…”

Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

“…At least for coronaviruses, it is known that members of the same virus family can use distinct pathways for fusion protein activation and virus entry. Whereas SARS-CoV and mouse hepatitis virus (MHV)-2 require uptake by endocytosis and subsequent proteolysis by cathepsins (Qiu et al, 2006;Simmons et al, 2005), infectious bronchitis virus only needs an acidification step (Chu et al, 2006) and the MHV-A59 strain or human CoV NL63 can enter the cell by pH-independent fusion at the plasma membrane (Huang et al, 2006;Qiu et al, 2006). The finding that the NiV F protein must be activated by endosomal proteases raised the possibility that NiV, similar to SARS-CoV, MHV-2 or Ebola virus, and in contrast to other paramyxoviruses, depends on pH-and CTSL-dependent mechanisms of entry.…”