Severe acute respiratory syndrome coronavirus replication is severely impaired by MG132 due to proteasome-independent inhibition of M-calpain.
The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5(-/-) mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.
Institute of Virology, Technische Universität München, Munich, Germany.
Martha
M
Schneider
Kerstin
K
Ackermann
Melissa
M
Stuart
Claudia
C
Wex
Ulrike
U
Protzer
Hermann M
HM
Schätzl
Sabine
S
Gilch
D000818:Animals; D001343:Autophagy / Q000187:drug effects; D000071187:Autophagy-Related Protein 5; D001483:Base Sequence; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D055785:Gene Knockdown Techniques; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000172:deficiency / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects* / Q000502:physiology*; D014709:Vero Cells; D053586:Virus Internalization / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects* / Q000502:physiology
C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D002154:Calpain; C449000:m-calpain; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde