a) Describe two ways the researcher could minimise experimenter bias in this study. b) Describe a way the researchers could minimise sample bias in this study. (distinct from your answers in part a) Nuclear import of SARS-CoV-2 nucleocapsid (N) protein is not inhibited by overmectin B.A. Loney* and M.A. Larkey* *Bundoora Institute for Applied Medical Research. Introduction The causative agent of the current COVID-19 pandemic, SARS-CoV-2, is a single stranded positive sense RNA virus that is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV). It has been previously shown that SARS-CoV-2 nucleocapsid (N) is present in the cytoplasm but can also actively localize to the nucleolus where it can interact with host proteins and also bind to viral RNA. It has also been previously shown that nuclear import of similar nucleocapsid proteins (including the HIV-1 nucleocapsid protein, NC) from other RNA viruses can be inhibited by the drug overmectin resulting in decreased viral replication efficiency. There are multiple nuclear import pathways mediated by different receptors. The two most common nuclear import pathways are mediated by the importin / heterodimer or by the importin homodimer. Overmectin has previously been demonstrated to inhibit nuclear import by disrupting the importin / pathway. In this study SARS-CoV-2 nucleocapsid (N) was transfected into Hela cells and the effectiveness of overmectin at inhibiting its nuclear import was determined. Methods Expression of N protein in the absence of other viral proteins. To investigate the nuclear import of SARS-CoV-2 N protein three constructs were created. 1. pEGFP-NCov2. The N gene (from SARS-CoV-2, isolate BJ04) was cloned into the eukaryotic expression vector pEGFP-C1 (Promega) such that expression of the N gene was under the control of a cytomegalovirus (CMV) polymerase Il promoter to express an N protein fusion with the C-terminal of EGFP. 2. pEGFP. The pEGFP-C1 expression vector alone. 3. EEGFP-TRF1. A positive control for nuclear import. The human TRF1 gene was cloned into the eukaryotic expression vector pEGFP-C1 to express an N protein fusion with the C-terminal of EGFP. TRF1 nuclear localisation has shown to be mediated by the importin homodimer. Hela cells were cultured in 12 separate culture plates at a density of 1 0 5 cells per 9.6 cm 2 plate with each plate containing 2 coverslips. Cells were cultured using Cell Biologics' Culture Complete Growth Medium with 5\% foetal calf serum at 3 7 C and 5% CO 2 . Cells were transfected with 2 g of either pEGFPNCov2 (plates 1,4,7 and 10), pEGFP (plates 2, 5, 8 and 11) or pEGFP-TRF1 (3, 6, 9 and 12) and 50 g of Lipofectamine (GibcoBRL). 12 hours post-transfection, even numbered plates were treated with 5 M overmectin in DMSO and odd numbered plates were left untreated. After 24 hours coverslips were removed, and the cells fixed. DAPI was added to visualise the nuclei and the localisation of GFP determined by fluorescent mic.

1. a) Describe two ways the researcher could minimise experimenter bias in this study.
b) Describe a way the researchers could minimise sample bias in this study. (distinct from
your answers in part a)
Nuclear import of SARS-CoV-2 nucleocapsid (N) protein is not inhibited by overmectin B.A.
Loney* and M.A. Larkey* *Bundoora Institute for Applied Medical Research. Introduction The
causative agent of the current COVID-19 pandemic, SARS-CoV-2, is a single stranded positive
sense RNA virus that is closely related to severe acute respiratory syndrome coronavirus (SARS-
CoV). It has been previously shown that SARS-CoV-2 nucleocapsid (N) is present in the
cytoplasm but can also actively localize to the nucleolus where it can interact with host proteins
and also bind to viral RNA. It has also been previously shown that nuclear import of similar
nucleocapsid proteins (including the HIV-1 nucleocapsid protein, NC) from other RNA viruses
can be inhibited by the drug overmectin resulting in decreased viral replication efficiency. There
are multiple nuclear import pathways mediated by different receptors. The two most common
nuclear import pathways are mediated by the importin / heterodimer or by the importin
homodimer. Overmectin has previously been demonstrated to inhibit nuclear import by
disrupting the importin / pathway. In this study SARS-CoV-2 nucleocapsid (N) was transfected
into Hela cells and the effectiveness of overmectin at inhibiting its nuclear import was
determined. Methods Expression of N protein in the absence of other viral proteins. To
investigate the nuclear import of SARS-CoV-2 N protein three constructs were created. 1.
pEGFP-NCov2. The N gene (from SARS-CoV-2, isolate BJ04) was cloned into the eukaryotic
expression vector pEGFP-C1 (Promega) such that expression of the N gene was under the
control of a cytomegalovirus (CMV) polymerase Il promoter to express an N protein fusion with
the C-terminal of EGFP. 2. pEGFP. The pEGFP-C1 expression vector alone. 3. EEGFP-TRF1. A
positive control for nuclear import. The human TRF1 gene was cloned into the eukaryotic
expression vector pEGFP-C1 to express an N protein fusion with the C-terminal of EGFP. TRF1
nuclear localisation has shown to be mediated by the importin homodimer. Hela cells were
cultured in 12 separate culture plates at a density of 1 0 5 cells per 9.6 cm 2 plate with each plate
containing 2 coverslips. Cells were cultured using Cell Biologics' Culture Complete Growth
Medium with 5% foetal calf serum at 3 7 C and 5% CO 2 . Cells were transfected with 2 g of
either pEGFPNCov2 (plates 1,4,7 and 10), pEGFP (plates 2, 5, 8 and 11) or pEGFP-TRF1 (3, 6,
9 and 12) and 50 g of Lipofectamine (GibcoBRL). 12 hours post-transfection, even numbered
plates were treated with 5 M overmectin in DMSO and odd numbered plates were left untreated.
After 24 hours coverslips were removed, and the cells fixed. DAPI was added to visualise the
nuclei and the localisation of GFP determined by fluorescent microscopy. 20 transfected cells
from each coverslip were chosen for analysis and the intensity of GFP fluorescence was
averaged for the nucleus (defined) by the DAPI staining and the cytoplasm. The remaining cells
in the culture dishes were collected and homogenised in RIPA buffer and the protein
concentration of the extracts determined by Lowry assay using BSA as a standard. 30 g of total
protein from each extract was electrophoresed on a 10% SDS-PAGE gel, transferred to a
nitrocellulose membrane and blocked in 5% skim milk in PBS. Membranes were subjected t
Western analysis using rabbit, anti-EGFP, anti-SARS-CoV-2 nucleocapsid protein and anti-
TRF1. followed by goat anti-rabbit IgG conjugated to horseradish peroxidase. Following each
incubation membranes were washed in PBS. Western blots were then developed using enhanced
chemiluminescence and exposed for 5 minutes to x -ray film. All three constructs (EGFP-

2. NCov2, EGFP-TRF1 and EGFP) were expressed in HeLa cells following transfection (Figure 1).
Transfection efficiency was between 55 and 65% for all of the constructs (data not shown). In
the control plates, EGFP remained localised in the cytoplasm while the EGFP-NCov2 was
predominantly localised to the nucleus (Figure 2). EGFPTRF1 also predominantly localised to
the nucleus. The addition of 5 M overmectin had no significant effect on the localisation of
EGFP-NCov2. Also as expected, 5 M overmectin had no significant effect on the localisation of
EGFP-TRF1 (Figure 3). These data demonstrate that the nuclear import of SARS-CoV-2
nucleocapsid protein is not mediated by the importin / heterodimer. Therefore, overmectin would
not be a suitable treatment for SARS-CoV-2 infection in humans.