Abstract
The host innate immune response is an important deterrent of severe viral infection in humans and animals. Nuclear import factors function as key gatekeepers that regulate the transport of innate immune regulatory cargo to the nucleus of cells to activate the antiviral response. Using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model, we demonstrate that SARS-COV ORF6 protein is localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, where it binds to and disrupts nuclear import complex formation by tethering karyopherin alpha 2 and karyopherin beta 1 to the membrane. Retention of import factors at the ER/Golgi membrane leads to a loss of STAT1 transport into the nucleus in response to interferon signaling, thus blocking the expression of STAT1-activated genes that establish an antiviral state. We mapped the region of ORF6, which binds karyopherin alpha 2, to the C terminus of ORF6 and show that mutations in the C terminus no longer bind karyopherin alpha 2 or block the nuclear import of STAT1. We also show that N-terminal deletions of karyopherin alpha 2 that no longer bind to karyopherin beta 1 still retain ORF6 binding activity but no longer block STAT1 nuclear import. Recombinant SARS-CoV lacking ORF6 did not tether karyopherin alpha 2 to the ER/Golgi membrane and allowed the import of the STAT1 complex into the nucleus. We discuss the likely implications of these data on SARS-CoV replication and pathogenesis.
Publication types
-
Research Support, N.I.H., Extramural
MeSH terms
-
Active Transport, Cell Nucleus / physiology
-
Animals
-
Caco-2 Cells
-
Cell Nucleus / genetics
-
Cell Nucleus / immunology
-
Cell Nucleus / metabolism*
-
Chlorocebus aethiops
-
Endoplasmic Reticulum / genetics
-
Endoplasmic Reticulum / immunology
-
Endoplasmic Reticulum / metabolism*
-
Gene Expression Regulation / genetics
-
Gene Expression Regulation / immunology
-
Golgi Apparatus / genetics
-
Golgi Apparatus / immunology
-
Golgi Apparatus / metabolism*
-
Humans
-
Immunity, Innate / genetics
-
Interferons / immunology
-
Interferons / metabolism
-
Mutation
-
Open Reading Frames / physiology*
-
Protein Structure, Tertiary / genetics
-
Recombinant Proteins / genetics
-
Recombinant Proteins / immunology
-
Recombinant Proteins / metabolism
-
STAT1 Transcription Factor / genetics
-
STAT1 Transcription Factor / immunology
-
STAT1 Transcription Factor / metabolism*
-
Severe acute respiratory syndrome-related coronavirus / genetics
-
Severe acute respiratory syndrome-related coronavirus / immunology
-
Severe acute respiratory syndrome-related coronavirus / metabolism*
-
Signal Transduction / genetics
-
Signal Transduction / immunology
-
Vero Cells
-
Virus Replication / physiology*
-
alpha Karyopherins / genetics
-
alpha Karyopherins / immunology
-
alpha Karyopherins / metabolism
-
beta Karyopherins / genetics
-
beta Karyopherins / immunology
-
beta Karyopherins / metabolism
Substances
-
KPNA2 protein, human
-
KPNB1 protein, human
-
Recombinant Proteins
-
STAT1 Transcription Factor
-
STAT1 protein, human
-
alpha Karyopherins
-
beta Karyopherins
-
Interferons