Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles - PubMed (original) (raw)

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles

Karin E Darpel et al. PLoS One. 2011.

Abstract

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. SDS-PAGE analysis of purified saliva from C. nubeculosus and C. sonorensis.

Purified saliva proteins (20 µg) from C. nubeculosus (lane 1) and C. sonorensis (lane 2) were analysed on 15% SDS-PAGE and visualized using silver staining. A representative figure of at least four separate and independent saliva collections is shown. A cluster of saliva proteins was identified between 40–70 kDa and 13–17 kDa molecular weight for both species. However C. sonorensis saliva has an additional prominent protein band at around 29 kDa (arrow), which was identified as “late trypsin” using mass spectrometry analysis. The presence of this 29 kDa protein in C. sonorensis saliva was consistent in 5 independent saliva collections.

Figure 2. Saliva proteins from C. sonorensis cleave VP2 of BTV-16 more efficiently than saliva proteins from C. nubeculosus.

Purified BTV-16 (RSAvvvv/16) virus particles (2 µg) were incubated for 3 hours at 37°C as a mock (lane 1) or with purified saliva proteins (2 µg) from either C. sonorensis or C. nubeculosus and BTV viral proteins were analyzed on a 12% SDS-PAGE using silver-staining for visualization. M represents the molecular weight markers. Lane 1: purified BTV-16 virus particles (2 µg); lane 2: purified BTV-16 virus particles incubated with C. sonorensis saliva proteins; lane 3: BTV-16 incubated with C. nubeculosus saliva proteins; lane 4: 2 µg of C. nubeculosus saliva proteins; lane 5: 2 µg of C. sonorensis saliva proteins. Outer capsid protein VP2 of BTV-16 (black arrow) was mostly cleaved by the C. sonorensis saliva proteins (lane 2), while only partial cleavage occurred with C. nubeculosus saliva proteins (lane 3). The cleavage of VP2 resulted in a cleavage product (CP) migrating at 110 kDa just underneath VP3. No further breakdown products of VP2 could be identified in these cleavage experiments. The red arrow indicates the 29 kDa trypsin like protein in C. sonorensis saliva and demonstrates its relative abundance compared to C. nubeculosus saliva. Figure 2 is a representative picture from 3 independent experiments.

Figure 3. C. sonorensis saliva or trypsin control cleave VP2 of BTV-1 particles, more efficiently than C. nubeculosus saliva.

Purified BTV-1 (RSArrrr/01) viral particles (2.5 µg) were incubated for 3 hours at 37°C either on its own as mock (lane 1) or with 0.5 or 1 µg saliva proteins from C. sonorensis susceptible (lanes 2 & 3), C. nubeculosus (lanes 5& 6) or C. sonorensis refractory (lanes 4&7) or with 1 or 0.5 µg trypsin as positive controls (lanes 8 & 9). M represents the molecular weight markers. Viral proteins were analysed by 10% SDS-PAGE and visualized by silver staining. BTV-1 VP2 protein (short arrow-lane 2) was completely cleaved by the saliva from both C. sonorensis strains (lanes 2&3 and 4&7) and by the control protease trypsin (lanes 8&9), resulting in two dominant cleavage products (CP) running at 110 kDa and 67 kDa respectively. Incubation with C. nubeculosus saliva proteins only resulted in partial cleavage of VP2 (lanes 5&6). VP2 cleavage products are either not detectable yet using 0.5 µg of C. nubeculosus saliva proteins (lane 5) or the smaller CP just starts to appear using 1 µg of C. nubeculosus saliva proteins. Due to a lower amount of saliva used many saliva proteins are not clearly identifiable on this SDS-PAGE. The 29 kDa sized protein identified as late trypsin (red arrow) is just visible in the lanes containing C. sonorensis saliva (lane 2,3,4 and 7).

Figure 4. Efficiency of BTV-1 VP2 cleavage by C. sonorensis saliva is temperature dependent.

BTV-1 particles (2 µg) were incubated with 1 µg of C. sonorensis saliva proteins at 4°C, 10°C, 15°C, 20°C, 25°C or 37°C for 2 hours in a final volume of 50 µl. Viral proteins were analysed on 10% SDS-PAGE and visualized using silver staining. Lane M shows molecular weight markers. Lane 1 and 8 are purified BTV-1 virus particles incubated at 37°C in the absence of proteases. After incubation at 4°C and 10°C (lane 2 and 3) intact BTV-1 VP2 was still visible, however some cleavage of VP2 had occurred resulting in intermediate cleavage products (ICP). At 15°C, no intact VP2 was visible but still only intermediate cleavage products (ICP) are generated. (lane 4). At higher temperatures, intermediate CP were completely cleaved to final CP (lane 7).

Figure 5. Comparison of the effect of different amounts of C. sonorensis saliva, trypsin and chymotrypsin.

Purified BTV-1 virus particles (2.5 µg) were incubate at 37°C for 30 minutes in a final volume of 50 µl using different amounts of C. sonorensis saliva proteins (S) , trypsin (T) and chymotrypsin (CT). BTV proteins were analyzed on a 10% SDS-PAGE using silver-staining for visualization. Lane 1: no proteases; lane 2: +2 µg S; lane 3: +1 µg S; lane 4: +0.5 µg S; lane 5: +1 µg T; lane 6: +0.5 µg T; lane 7: +0.1 µg T; lane 8: +0.05 µg T; lane 9: +0.05 µg CT; lane 10: +0.01 µg CT: lane 11: +0.005 µg CT; lane 12: +0.001 µg CT. The same levels of BTV-1 VP2 cleavage were obtained with 2.0 µg of C. sonorensis saliva proteins, 0.5 µg of trypsin or an estimated 0.008 µg of chymotrypsin. For all three different proteases, C. sonorensis saliva, trypsin or chymotrypsin it can be seen that using lower amounts of proteases generates intermediate cleavage products (ICP) (e.g. lane 3 and 4, lane 7 and 8 and lane 12) which are further cleaved using higher amounts of proteases, leading to the two final VP2 cleavage products (CP) migrating at ∼110 kDa and ∼67 kDa respectively (e.g. lane 2, lane 5 and lane 9 and 10).

Figure 6. The ability of Culicoides saliva to cleave EHDV VP2 depends on the strain of EHDV.

Purified EHDV-2 virus particles (1 µg) from two different strains, either CAN1962/01 or AUS1979/05, were incubated in a final volume of 50 µl for 3 hours at 37°C with or without saliva proteins (1 µg) from either C. sonorensis or C. nubeculosus. Viral proteins were analysed on 12% SDS-PAGE and visualised using silver staining. Lane M indicates molecular weight markers. Lane 1: CAN1962/01 virus particles; lane 2: CAN1962/01 virus particles with C. sonorensis saliva proteins; lane 3: CAN1962/01 virus particles with C. nubeculosus saliva proteins; lane 4: AUS1979/05 virus particles; lane 5: AUS1979/05 virus particles with C. sonorensis saliva proteins; lane 6: AUS1979/05 virus particles with C. nubeculosus saliva proteins. EHDV VP2 of the AUS1979/05 strain (black arrow) was cleaved by the saliva proteins from either Culicoides species and intact VP2 was not detected (lanes 5 & 6). In contrast EHDV VP2 of strain CAN1962/01 (white arrow) was still intact in the presence of saliva proteins from either Culicoides species (lanes 2 and 3). The black and white arrows indicate the VP2 proteins, which migrate at different size for the two different EHDV-2 strains. The red arrows highlight the 29 kD protein identified as late trypsin in the C. sonorensis saliva.

Figure 7. The effect of different protease inhibitors on C. sonorensis saliva, trypsin or chymotrypsin.

BTV-1 purified virus particles were incubated with different amounts of C. sonorensis saliva proteins (S), trypsin (T) or chymotrypsin (CT) in a final volume of 50 µl for 30 minutes at 37°C, in the presence or absence of the following protease inhibitors : Bowman-Birk serine protease inhibitor (BBI); α-chymotrypsin inhibitor (CTInh) and Trypsin Inhibitor SII (Tinh). The viral proteins were analysed on 10% SDS-PAGE and visualized using silver staining. Lane M indicates molecular weight markers. Each reaction contained 2.5 µg BTV-1 particles with the different additions listed for each lane. Panel A: lane 1: mock treated virus particles, lane 2: +1 µg S, lane 3: +1 µg S and 1 µg BBI, lane 4: +1 µg S and 20 µg BBI, lane 5: +0.05 µg CT, lane 6: +0.05 µg CT and 1 µg BBI, lane 7: +0.05 µg CT and 20 µg BBI, lane 8: +0.5 µg T, lane 9: +0.5 µg T and 1 µg BBI. (Picture generated from one gel, not all lanes shown). Cleavage of BTV-1 VP2 by all three different proteases is prevented in the presence of the serine protease inhibitor, however C. sonorensis saliva proteins and chymotrypsin need a higher concentration of inhibitor to completely inhibit protease activity. Panel B: lane 1: mock treated virus particles, lane 2: +0.5 µg T, lane 3: +1 µg S, lane 4: +0.5 µg CT, lane 5: mock treated virus particles, lane 6: +0.5 µg T and 30 µg CTInh, lane 7: +1 µg S and 30 µg CTInh, lane 8: +0.5 µg CT and 30 µg CTInh, lane 9: +0.01 µg T and 30 µg CTInh, lane 10: +0.05 µg T and 30 µg CTInh, lane 11: +0.01 µg T; lane 12: +0.05 µg T. (Picture generated from two gels, not all lanes shown). The α-chymotrypsin inhibitor does not inhibit the cleavage of BTV-1 VP2 by trypsin but has a partial inhibition effect on the cleavage of VP2 by C. sonorensis saliva proteins. Panel C: lane 1: mock treated virus particles, lane 2: +0.5 µg T; lane 3: +1 µg S ; lane 4: mock treated virus particles, lane 5:+0.5 µg T and 2 µg TInh, lane 6: +1 µg S and 2 µg TInh, lane 7: +1 µg S and 4 µg TInh, lane 8: mock treated virus particles, lane 9: +0.05 µg CT, lane 10: +0.05 µg CT and 2 µg TInh (Picture generated from two gels not all lanes shown). Cleavage of BTV-1 VP2 by trypsin is completely inhibited by the trypsin inhibitor, however the protease activity of C. sonorensis saliva is only partially inhibited.

Figure 8. Infectivity of purified BTV-1 particles for BHK and KC cells following incubation with C. sonorensis saliva, trypsin or chymotrypsin.

Panel A: BTV-1 purified particles (2.5 µg) were incubated either in the absence of proteases, or with 5, 10 or 15 µg of C. sonorensis saliva proteins, in a final volume of 100 µl for 3 hours at 37°C. Panel B: BTV-1 purified particles (2.5 µg) were incubated either in the absence of proteases or with 1 or 5 µg of trypsin (T), in a final volume of 100 µl for 3 hours at 37°C. Panel C: BTV-1 purified particles (2.5 µg) were incubated either in the absence of proteases or with 0.5, 1 or 5 µg of chymotrypsin (CT), in a final volume of 100 µl for 3 hours at 37°C. The treated BTV-1 particles were added to plain media (GMEM) and Log10 dilutions from −1 to −11 were prepared. These dilutions were titrated in either BHK cells or KC cells in 96 well plates (50 µl/ well and 8 wells/ dilution) and incubated at 37°C or 30°C respectively. The BHK cells were examined for CPE on day 5 and 7 post inoculation. The KC cells were ‘back titrated’ onto new BHK cell plates 7 d.p.i. and the titre determined by reading CPE on day 5 and 7 post transfer. Each data point represents at least 3–6 individual experiments (standard deviation = error bar). Cleavage of BTV-1 VP2 by different proteases increased the infectivity of modified particles for KC cells by 10 to 12 fold, while it reduced the infectivity for BHK cells between 2 and >20 fold.

References

1. Ribeiro JMC. Blood-Feeding Arthropods: Live Syringes or Invertebrate Pharmacologists? Infectious Agents and Disease. 1995;4:143–152. -PubMed
1. Schneider BS, Soong L, Girard YA, Campbell G, Mason P, et al. Potentiation of West Nile encephalitis by mosquito feeding. Viral Immunology. 2006;19:74–82. -PubMed
1. Schneider BS, Soong L, Zeidner NS, Higgs S. Aedes aegypti salivary gland extracts modulate anti-viral and T(H)1/T(H)2 cytokine responses to Sindbis virus infection. Viral Immunology. 2004;17:565–573. -PubMed
1. Edwards JF, Higgs S, Beaty BJ. Mosquito feeding-induced enhancement of Cache Valley virus (Bunyaviridae) infection in mice. Journal of Medical Entomology. 1998;35:261–265. -PubMed
1. Limesand KH, Higgs S, Pearson LD, Beaty BJ. Potentiation of vesicular stomatitis New Jersey virus infection in mice by mosquito saliva. Parasite Immunology. 2000;22:461–467. -PubMed

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles - PubMed (original) (raw)