Detection of a fourth orbivirus non-structural protein

Abstract

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1-VP7) and 3 non-structural proteins (NS1-NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.

Figure 1. Synonymous site conservation in BTV VP6 coding sequence.

Comparison of 67 BTV Seg-9 sequences. Panels 1 and 2 show the positions in the aligned sequences, of stop codons (blue triangles) in the +1 and +2 reading frames relative to the VP6 reading frame, and alignment gaps (green rectangles). Note the conserved absence of stop codons in the +1 frame in the region corresponding to the NS4 ORF. The vertical red line in panel 1 indicates the location of a completely conserved +1 frame AUG codon. One sequence (out of 67) with premature termination codons (PTC) within the NS4 ORF is indicated. Panel 3 shows the probability that the degree of conservation within a given window could be obtained under a null model of neutral evolution of VP6-frame synonymous sites. Panel 4 shows the level of conservation represented by the ratio of the observed number of substitutions within a given window, to the number expected under the null model.

Figure 2. Recombinant NS4 proteins of BTV and GIV.

NS4 of BTV and GIV were expressed as soluble GST fusion proteins in C41 bacteria, purified as described in Materials and Methods, then analysed by SDS-PAGE and stained with Coomassie blue. M indicates the molecular weight marker, labelled in kDa.

Figure 3. Western blot analysis of BTV-8 in KC cells.

BTV-8 infected KC cell lysates analysed by SDS PAGE/Western blot using anti-BTV NS4 antibodies. BTV_KC7 = KC cells harvested at 7 days post-infection. M indicates the molecular weight marker, labelled in kDa. Lane NI indicates non-infected KC cells which do not show any cross reactivity of anti-BTV NS4 antibody and cellular proteins. NS4 that was identified in infected cells using anti-NS4 antibodies (∼12 kDa) was absent from non-infected cells.

Figure 4. Western blot analysis of BTV-8 in BHK-21 cells.

BTV-8 infected BHK-21 cell lysates analysed by SDS PAGE/Western blot using anti-BTV NS4 antibodies. M indicates the molecular weight marker, labelled in kDa. BTV_BHK24 = BHK-21 cells harvested at 24 hours hours post-infection, respectively. Lane NI indicates non-infected BHK-21 cells which do not show any cross reactivity of anti-BTV NS4 antibody and cellular proteins. NS4 that was identified in infected cells using anti-NS4 antibodies (∼12 kDa) was absent from non-infected cells.

Figure 5. Western blot analysis of GIV in BHK-21 cells.

GIV infected BHK-21 cell lysates analysed by SDS PAGE/Western blot using anti-GIV NS4 antibodies. Lane GIV_BHK24 = BHK-21 cells harvested at 24 hours post-infection. Lane M indicates the molecular weight marker, labelled in kDa. Lane NI indicates non-infected BHK-21 cells which do not show any cross reactivity of anti-GIV NS4 antibody and cellular proteins. A protein was identified by the anti-GIV NS4 antibody in infected cells (approximately 20 kDa) that is absent from non-infected cells.

Figure 6. Western blot analysis of purified BTV-8.

A: SDS-PAGE of purified BTV-8 showing all seven structural proteins stained with Coomassie blue (note the absence of a detectable band of the appropriate size for NS4). B: western blot analysis using purified BTV-8 virus particles (as shown in panel D) probed with anti-NS4 antibodies. The reaction is negative, indicating that NS4 is truly non-structural. Lane M: molecular weight markers, labelled in kDa. Lane V: the structural proteins of purified BTV-8 virions are indicated.

Figure 7. Western blot analysis of BTV-8 infected and non-infected BHK-21 cells using anti-VP2 antibodies.

Non-infected (lane N-INF) and BTV-8 infected cells (lane INF) probed with anti-BTV-8 VP2 antibodies raised in mice against recombinant VP2. The antiserum did not cross react with non-infected cells and identified a protein of approximately 110 kDa in infected cells (corresponds to the theoretical size of VP2).

Figure 8. Western blot analysis of BTV-8 infected and non-infected BHK-21 cells using anti-BTV-8 antibodies.

Non-infected (lane N-INF) and BTV-8 infected cells (lane INF) probed with anti-BTV-8 immune serum from infected mice. The antiserum did not cross react with non-infected cells and identified several viral proteins in infected cells. Lane M represents the marker labelled in kDa.

Figure 9. Western blot analysis of the nuclear fraction from BTV-8 infected BHK-21 cells.

The nuclear fraction was prepared as described under materials and methods. Infected and non-infected cells were used for the assay. The extracts were analysed by SDS-PAGE/Western blot. Anti-BTV NS4 antibodies identified a protein in the nuclear fraction of infected cells (NF_in, indicated by an arrow) which is absent from the nuclear fraction of non-infected cells (NF_nin).

Figure 10. Distribution of NS4 in BTV-8 and GIV infected BHK-21 cells at 4 hours post-infection.

A: BHK-21 cells infected with BTV-8 showing NS4 mainly in the cytoplasm. B: BHK-21 cells infected with GIV showing NS4 both in the cytoplasm. Cells were incubated with anti-BTV-8 NS4, or anti-GIV NS4 rabbit antibodies and anti-alpha tubulin mouse antibodies. Cells were then incubated with Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG and Alexa Fluor 568 (red fluorescence) conjugated anti-mouse.

Figure 11. Distribution of NS4 in BTV-8 and GIV infected BHK-21 cells at 24 hours post-infection.

A: BHK-21 cells infected with BTV-8 showing NS4 both in the cytoplasm and nucleus. B: BHK-21 cells infected with GIV showing NS4 both in the cytoplasm and nucleus. Cells were incubated with anti-BTV-8 NS4, or anti-GIV NS4 rabbit antibodies and anti-alpha tubulin mouse antibodies. Cells were then incubated with Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG and Alexa Fluor 568 (red fluorescence) conjugated anti-mouse.

Figure 12. Distribution of NS4 in BTV-8 and GIV infected BHK-21 cells at 72 hours post-infection.

A: BHK-21 cells infected with GIV showing fluorescence in the cytoplasm and cell membrane but less in the nucleus. B: BHK-21 cells infected with BTV-8 showing fluorescence in the cytoplasm and cell membrane but less in the nucleus. Cells were incubated with anti-BTV-8 NS4, or anti-GIV NS4 rabbit antibodies. Cells were then incubated with Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG.

Figure 13. BTV-8 infected and non-infected BHK-21.

A: BTV-8 infected BHK-21 cells at 36 hours pi, showing cells at different stages of infection. This panel shows a cell (bottom of the panel) with depleted tubulin and an accumulation of the NS4 in the cytoplasm and to a much lesser extent in the nucleus. The panel show cells with a less advanced infection (top) with lower expression of NS4 and an intact alpha-tubulin network. B: Non-infected BHK-21 cells stained with DAPI, anti-alpha-tubulin and anti-NS4 antibodies. Cells were incubated with anti-BTV-8 NS4 rabbit antibodies and anti-alpha tubulin mouse antibodies. Cells were then incubated with Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG and Alexa Fluor 568 (red fluorescence) conjugated anti-mouse.

Figure 14. BTV-8 infected BHK-21 showing fluorescence in the nucleoli.

A: fluorescence signal using anti-NS4 antibodies showing the NS4 in the cytoplasm and nucleus. B: Fluorescence signal using anti-NS4 antibodies overlaid onto cells imaged by differential interference contrast showing fluorescence around the nucleus in the cytoplasm and green fluorescence indicated by an arrow overlaid onto the nucleolus. Cells were incubated with anti-BTV-8 NS4 rabbit antibodies and then incubated with Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG.

Figure 15. Co-localisation of NS4 and fibrillarin in BTV-8 infected BHK-21.

Confocal image of cells infected with BTV-8 showing fibrillarin (A: in red) detected by anti-fibrillarin antibody (Serotech), NS4 (B: in green) identified by anti-BTV-8 NS4 antibodies, nuclei stained blue with DAPI (C) and a merge of these 3 subsets (D) showing co-localisation of the BTV-8 NS4 and the fibrillarin (yellow).

Figure 16. Confocal fluorescence microscopy of BHK-21 cells expressing NS4 of BTV-8.

Cells were transfected with pCI-BTVNS4 expressing NS4 of BTV-8. At 48 hours post-transfection, cells were fixed with paraformaldehyde, permeabilised with 0.1% Triton X-100 and incubated with anti-BTV-8 NS4 antibodies. Cells were then incubated with Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG (Invitrogen). A: a focal plane of BHK-21 cells expressing NS4 and showing cytoplamsic fluorescence. In a large number of cells, NS4 was found to form spherical bodies (as shown in the figure) having a diameter between 0.7 and 1 µm. B: a focal plane of BHK-21 cells showing both cytoplasmic and nucleolar (indicated by arrows) fluorescence. Similar results were obtained with cells transfected with pCI-GIVNS4 expressing NS4 of GIV.

Figure 17. Confocal fluorescence microscopy of GIV or BTV-8 infected BHK-21 cells.

A: cells were infected with GIV and show spherical bodies (identified by anti-GIV NS4 antibodies) similar to those identified in cells transfected with pCI-BTVNS4. B: identification of the spherical bodies (in BTV-8 infected cells) as lipid droplets, by staining with the lipid stain oil-red-O. B-1: cells stained with oil-red-O. B-2: cells probed with anti-BTV NS4 antibodies. B-3: cells stained with DAPI. B-4: Co-localisation of NS4 with lipid droplets; oil-red-O stains the lipid droplet in red while the green fluorescence surrounding the lipids indicates BTV NS4.

Figure 18. Co-localisation of GIV NS4 or BTV NS4 with lipid droplets in BHK-21 cells.

A: cells transfected with pCI-GIVNS4 stained with oil-red-O and probed with anti-GIV NS4 antibodies. F: Cells infected with BTV-8 stained with oil-red-O and probed with anti-BTV NS4 antibodies.

Figure 19. Non-infected BHK-21 cell stained with oil-red-O and anti-BTV NS4.

Figure 20. Western blot analysis BHK-21 cells transfected with plasmid pCI-BTVNS4.

Cells were transfected with pCI-BTVNS4 expressing NS4 of BTV-8. At 48 hours post-transfection cells were scraped, lysed in sample denaturation buffer and analysed by SDS-PAGE/Western blotting, using BTV-8 immune serum from experimentally infected mice as primary antibody. M indicates the molecular weight marker, labelled in kDa. Lane labelled as pCI-BTVNS4 indicates cells transfected with pCI-BTVNS4 plasmid, NT indicates non-transfected cells. NS4 that was identified in transfected cells using anti-NS4 antibodies (∼12 kDa) was absent from non-transfected cells.

Figure 21. Dicer competition assay.

Lane RL: dsRNA ladder labelled in base pairs. Lane 1: dsRNA ladder pre-incubated with GIV NS4 followed by Dicer. GIV NS4 prevented cleavage by Dicer. Lane 2: dsRNA ladder pre-incubated with BTV-8 NS4, followed by Dicer. BTV-8 NS4 did not prevented Dicer from cleaving long dsRNAs into 21 bp long siRNAs. Lane 3: ladder incubated with Dicer as a positive digestion-control. Lane 4: dsRNA ladder pre-incubated with VP9 of BAV followed by Dicer. VP9 of BAV did not prevent Dicer from cleaving long dsRNAs into 21 bp long siRNA. Lane 5: dsRNA ladder incubated with VP9 of BAV. VP9 of BAV did not affect the integrity of dsRNA.

Figure 22. DNase I competition assay.

Lane DL: dsDNA ladder labelled in base pairs. Lane 1: dsDNA ladder pre-incubated with GIV NS4 followed by DNase I. GIV NS4 protected the ladder against DNase cleavage. Lane 2: dsDNA ladder pre-incubated with BTV-8 NS4, followed by DNase I, showing that BTV NS4 protected against DNase cleavage. Lane 3: Ladder incubated with DNase I as positive control of digestion. Lane 4: dsDNA ladder pre-incubated with VP9 of BAV followed by DNAse I. VP9 of BAV did not prevent DNAse I from degrading dsDNA. Lane 5: dsDNA ladder incubated with VP9 of BAV. VP9 of BAV did not affect the integrity of dsDNA.

Figure 23. Colorimetric assay to detect interactions of NS4 with dsRNA.

The graph shows colorimetric OD readings plotted against concentrations of dsRNA. Increasing concentrations (from 1 to 640 ng) of a biotinylated dsRNA were bound to wells coated with streptavidin. BTV NS4 or GIV NS4 were added to the wells in triplicate. Wells not containing dsRNA/NS4 were included as negative controls. Wells from which dsRNA was omitted, but in which NS4 (BTV or GIV) alone was incubated were also included as controls. Only wells containing the dsRNA to which GIV NS4 was added reacted with anti-GIV antibodies as indicated by increasing OD readings. The readings were almost linear (reaching a plateau at 320 ng of dsRNA) indicating that dsRNA acted as a target for binding of GIV NS4.

Figure 24. Hydrophobicity profiles of orbivirus NS4.

Superimposed hydrophobicity profiles based on a Clustal X generated alignment of orbivirus NS4 amino acid sequences. The residue numbers are relative to NS4 of GIV (the longest NS4 identified to date). GIV NS4 (dashed line), BTV NS4 (blue line), AHSV NS4 (red line), YUOV NS4 (green line) and PHSV NS4 (purple line). The plots show significant similarities (particularly between residues 40–60), with GRAVY values of −1.02 to −1.05, except GIV NS4 which had a GRAVY value of −0.448.