Abstract Background
The
ORF6 protein is one of the eight accessory proteins of the severe acute
respiratory syndrome coronavirus (SARS-CoV). Numerous properties of
ORF6 have been documented and this study focuses on two of these,
namely, its ability to suppress the expression of co-transfected
expression constructs and its subcellular localization to vesicular
structures
Conclusions
By
combining quantitative real-time PCR and transient transfection system,
a simple and safe method is established to measure ORF6's ability to
suppress the expression of co-transfected myc-nsp8. In addition,
immunofluorescence analysis revealed that the subcellular localization
of ORF6 when expressed on its own is similar to that observed in
SARS-CoV infected cells. Through the use of these two assays, a putative
diacidic motif in the ORF6 protein was found to influence its
subcellular localization and ability to suppress the expression of
co-transfected expression constructs.
Go to: Background
An
outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003 which
carried with it a fatality rate of 8% was traced to a novel coronavirus
dubbed the SARS Coronavirus (SARS-CoV). This novel coronavirus was
eventually classified as a Group IIb coronavirus, a subset of the Group
II coronaviruses. The subclassification was, in part, due to the
presence of several accessory genes in the coronavirus which have no
known homologs within the family Coronaviridae. These accessory
genes have been the subject of study by many groups (for reviews, see 1
and 2) and have been assigned a plethora of physical characteristics
and intracellular functions. Most importantly, almost all of these
accessory genes have been shown to be dispensable for viral replication
in cell culture, with the exception of the 3a accessory gene (3). It has
been suggested these accessory genes have subtle effects on SARS-CoV
replication and may be more important for viral replication or
pathogenesis in vivo.
- One of these accessory genes, ORF6, encodes for a ~7kDa protein with a hydrophobic N-terminal and that has been suggested to have a N-endo-C-endo conformation (4).
- Several groups have undertaken to characterize the protein product of the ORF6 gene and found that it interacts with the nsp8 protein from the SARS replicase complex (5),
- is able to increase infection titer during early infection at low multiplicity of infection (6),
- increase the rate of cellular gene synthesis (7),
- inhibit interferon production (8),
- and inhibit the nuclear translocation of STAT1 by interacting with karyopherin α2 (9).
- Most recently, the ORF6 protein has been suggested to induce intracellular membrane rearrangements resulting in a vesicular population in the infected cell which could possibly serve some role in increasing replication (10).
Such virus-induced or virus associated
vesicles have previously been shown in other viral infections, such as
protein trafficking in Herpes simplex virus (11) and Sendai virus (12).
Members of the coronavirus family have been shown by several groups to
also utilize vesicular structures within the infected cell; most of
these studies suggest that vesicles play a role in viral replication
(13-16).
We have previously shown that
the ORF6 protein colocalizes with Lamp1-positive vesicles in SARS-CoV
infection (5) and also with the nsp8 protein in the same set of infected
cells, indicating a possible role for the ORF6 protein in the
replicative process of SARS-CoV.
However, there has been, to date,
little work done to link the subcellular localization of the ORF6
protein to its known functions. Gallagher and co-workers have shown that
the ability of ORF6 to impede nuclear translocation resulted in the
suppression of expression of transgenes from co-transfected expression
constructs if these need transcription in the nucleus (17). Using this
knowledge, a simple co-transfection system and alanine substitution
mutants in the ORF6 gene are used here to determine regions of the ORF6
protein that modulate its ability to suppress the expression of
co-transfected myc-nsp8. The subcellular localization of these mutants
is then examined by immunofluorescence experiments and compared to that
of wildtype ORF6.
(Kommentti: ORF-kaavassa näyttää nCoV 2019 omaavat samantapaisen ORF 6 :n)
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