Phenoloxidase activity acts as a mosquito innate immune response against infection with semliki forest virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses

Spread of SFV in mosquito and vertebrate cells.

<p>(<b>A</b>) The addition of glutathione (GSH) to medium enhances the spread of SFV. U4.4 cells were infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F) or SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R) at an MOI of 0.005 followed by determination of <i>FFLuc</i> activity at 48 h p.i. + GSH: 0.5 mM GSH; − GSH: negative control. Each bar represents the mean from triplicate cultures; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>B</b>) Egf1.0 has no effect on SFV spread in BHK-21 cells. Cells were infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F) or SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R) at an MOI of 0.005 followed by determination of <i>FFLuc</i> activity at 24 h and 48 h p.i. Each bar represents the mean from triplicate cultures; error bars show standard deviation. This experiment was repeated three times with similar results.</p

Recombinant SFV expresses Egf1.0 and inhibits PO activity in U4.4 cell-conditioned medium.

<p>(<b>A</b>) Immunoblots showing Egf1.0 expression and secretion from mosquito cells. U4.4 cells were infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0F or SFV4(3F)-<i>ZsGreen</i>-Egf1.0R at an MOI of 10 followed by preparation of cell lysate and medium samples at 48 h p.i. as indicated in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#s4" target="_blank">Materials and Methods</a>. The left blot was probed with an anti-SFV nsP3 antibody with individual lanes labeled as follows: U4.4 cells infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0R (R = cell lysate, Rm = conditioned medium), U4.4 cells infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0F (F = cell lysate, Fm = conditioned medium), or uninfected cells (U = cell lysate, Um = conditioned medium). Black star identifies the nsP3-ZsGreen protein, only detected in lysates from SFV-infected cells. Black diamond indicates bovine serum albumin (non-specifically detected because of high abundance). The right blot shows the same samples probed with an anti-Egf1.0 antibody. A control lane (C) was added to this blot (purified, recombinant Egf1.0). Note that Egf1.0 is only detected in the control lane, and F and Fm lanes. Black arrow indicates uncut Egf1.0; open arrow identifies a band corresponding to the predicted C-terminal domain of Egf1.0 after PAP cleavage. Molecular mass markers indicated to the left. (<b>B</b>) PO activity in conditioned medium from uninfected U4.4 cells (Control), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F), SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F with <i>E. coli</i> added to the medium (Egf1.0F+<i>E. coli</i>), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0R with <i>E. coli</i> added to the medium (Egf1.0R+<i>E. coli</i>), or medium from uninfected cells with <i>E. coli</i> added (<i>E. coli</i>). PO activity was measured as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#ppat-1002977-g002" target="_blank">Fig. 2A</a>; 1 ml of conditioned medium was taken at 48 h p.i. from 2.6×10⁵ U4.4 cells infected at an MOI of 10, or uninfected (Control). Each bar represents the mean from 10 reactions; error bars show standard deviation. This experiment was repeated three times with similar results.</p

Viruses used in study.

<p>(<b>A</b>) SFV (prototype strain SFV4). (<b>B</b>) SFV(3H)-<i>FFLuc</i>-Egf1.0F and SFV(3H)-<i>FFLuc</i>-Egf1.0R, encoding Firefly luciferase (<i>FFLuc</i>) as part of the non-structural polyprotein (inserted between duplicated nsP2 cleavage sites at the nsP3/4 junction), and from a duplicated subgenomic promoter the melanisation inhibitor Egf1.0 in sense (F virus; top) or (as negative control) antisense orientation (R virus; bottom). (<b>C</b>) SFV(3F)-<i>ZsGreen</i>-Egf1.0F and SFV(3F)-<i>ZsGreen</i>-Egf1.0R, expressing ZsGreen inserted into the C-terminal region of nsP3, and from a duplicated subgenomic promoter the melanisation inhibitor Egf1.0 in sense (F virus; top) or (as negative control) antisense orientation (R virus; bottom).</p