Monitoring of S protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus.
Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, a fatal pulmonary disorder with no effective treatment. We found that SARS-CoV spike glycoprotein (S protein), a key molecule for viral entry, binds to calnexin, a molecular chaperone in the endoplasmic reticulum (ER), but not to calreticulin, a homolog of calnexin. Calnexin bound to most truncated mutants of S protein, and S protein bound to all mutants of calnexin. Pseudotyped virus carrying S protein (S-pseudovirus) produced by human cells that were treated with small interfering RNA (siRNA) for calnexin expression (calnexin siRNA-treated cells) showed significantly lower infectivity than S-pseudoviruses produced by untreated and control siRNA-treated cells. S-pseudovirus produced by calnexin siRNA-treated cells contained S protein modified with N-glycan side chains differently from other two S proteins and consisted of two kinds of viral particles: those of normal density with little S protein and those of high density with abundant S protein. Treatment with peptide-N-glycosidase F (PNGase F), which removes all types of N-glycan side chains from glycoproteins, eliminated the infectivity of S-pseudovirus. S-pseudovirus and SARS-CoV produced in the presence of α-glucosidase inhibitors, which disrupt the interaction between calnexin and its substrates, showed significantly lower infectivity than each virus produced in the absence of those compounds. In S-pseudovirus, the incorporation of S protein into viral particles was obviously inhibited. In SARS-CoV, viral production was obviously inhibited. These findings demonstrated that calnexin strictly monitors the maturation of S protein by its direct binding, resulting in conferring infectivity on SARS-CoV.
Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan. mfukushi@hiroshima-u.ac.jp
Masaya
M
Fukushi
Yoshiyuki
Y
Yoshinaka
Yusuke
Y
Matsuoka
Seisuke
S
Hatakeyama
Yukihito
Y
Ishizaka
Teruo
T
Kirikae
Takehiko
T
Sasazuki
Tohru
T
Miyoshi-Akiyama
D000818:Animals; D037281:Calnexin / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D045473:Severe acute respiratory syndrome-related coronavirus / Q000472:pathogenicity / Q000502:physiology*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014779:Virus Replication
D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D037281:Calnexin