Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins.
The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nsp12 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.
Department of Microbiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
Erik
E
Prentice
Josephine
J
McAuliffe
Xiaotao
X
Lu
Kanta
K
Subbarao
Mark R
MR
Denison
D000818:Animals; D000914:Antibodies, Viral; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D006517:Murine hepatitis virus / Q000201:enzymology / Q000235:genetics; D016366:Open Reading Frames; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D000914:Antibodies, Viral; D004279:DNA, Viral; D000939:Epitopes; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase