Inhibition of Orbivirus Replication by Fluvastatin and Identification of the Key Elements of the Mevalonate Pathway Involved

Abstract

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus), dengue virus (Flavivirus), African swine fever virus (Asfarviridae) and poliovirus (Picornaviridae). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR^(-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Keywords: BTV; HMG-CoA reductase; IFNAR(−/−) mice; antiviral; bluetongue virus; fluvastatin; orbivirus.

Figure 1

A neighbour-joining phylogenetic tree constructed using MEGA X, showing the clustering of Culicoides sonorensis 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase amongst insect HMG-CoA reductase enzymes. Accession numbers of sequences used to construct the tree are provided in Supplemental Table S1.

Figure 2

Effect of fluvastatin on cell viability. (A) Effect of increasing concentrations of fluvastatin on BSR cell viability. Cells were harvested at 72 h post-treatment and assessed by an MTT viability assay. (B) Effect of increasing concentrations of fluvastatin on KC cell viability. Cells were harvested on day 5 post-treatment.

Figure 3

The mevalonate pathway of mammals. Key steps inhibited by statins (inhibition of HMG-CoA reductase), zaragozic acid A (inhibition of squalene synthase), geranyl-geranyl pyrophosphate or farnesyl pyrophosphate inhibitors are indicated.

Figure 4

YFV17D-infected BSR cells treated with fluvastatin, inhibitors of geranyl-geranylation (geranyl-geranyl transferase inhibitor GGTI-2133 or farnesyl pyrophosphate transferase inhibitor FTPIII), inhibitors of squalene synthase (zaragozic acid), or treated with fluvastatin then supplemented with components of the mevalonate pathway (mevalonic acid, geranyl-geranyl pyrophosphate, farnesyl pyrophosphate or cholesterol) in attempts to restore virus replication in fluvastatin-treated cells. These results are representative of four distinct experiments. Error bars with standard deviations are shown.

Figure 5

Confocal fluorescence microscopy of Great Island virus (GIV) or BTV infected BSR cells treated with fluvastatin. GIV infected cells were labelled with anti-GIV-NS4 (non-structural protein) and Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG. BTV infected cells were labelled with anti-BTV-NS4 and Alexa Fluor 488 (green fluorescence) conjugated anti-rabbit IgG or anti-BTV-8 VP2 and Alexa Fluor 568 (red fluorescence) conjugated anti-mouse IgG. Nuclei were stained with DAPI (blue). (A) Non-treated GIV infected cell control showing the NS4 spherical bodies. (B) GIV infected cells treated with fluvastatin, showing an almost complete disappearance of the NS4 spherical bodies and low levels of NS4 expression in the cells. (C) Non-treated BTV-8 infected-cell control showing NS4 in the cytoplasm and nucleus. (D) BTV infected cells treated with fluvastatin, showing an almost complete disappearance of the NS4 signal. (E) Non-treated BTV-8 infected-cell control showing strong expression of VP2 across the cells. (F) BTV-8 infected cells treated with fluvastatin, showing a significant drop of the VP2 signal.

Figure 6

Effects of fluvastatin, mevalonate pathway-components and inhibitors on BTV replication in BSR cells. BTV-8 titres (PFU/mL) generated in BTV-8-infected BSR cells, treated with: fluvastatin; inhibitors of geranyl-geranylation (geranyl-geranyl transferase inhibitor GGTI-2133, or farnesyl pyrophosphate transferase inhibitor FTPIII); or inhibitors of squalene synthase (zaragozic acid A). Some cells were treated with fluvastatin then supplemented with components of the mevalonate pathway (mevalonic acid, geranylgeranyl pyrophosphate, farnesyl pyrophosphate or cholesterol), to inhibit, or in attempts to restore, virus replication in fluvastatin-treated cells. These results are representative of 4 independent experiments. Error bars with standard deviations are shown.

Figure 7

Effects of fluvastatin, mevalonate pathway inhibitors and components on BTV replication in KC cells. BTV-8 virus titres (PFU/mL) generated by infected KC cells treated with fluvastatin, inhibitors of geranylgeranylation (geranyl-geranyl transferase inhibitor GGTI-2133 and farnesyl pyrophosphate transferase inhibitor FTPIII), or inhibitors of squalene synthase (zaragozic acid A). Some cells were treated with fluvastatin then supplemented with mevalonic acid in attempts restore virus replication. These results are representative of three independent experiments. Error bars with standard deviations are shown.

Figure 8

Survival curves of IFNAR^(−/−) mice.