TLR ligand induced anti-HIV-1 response blocks infection prior to reverse transcription.

<p>MDM were treated with TLR ligands as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024193#s4" target="_blank">Materials and Methods</a> prior to and during exposure to ADA. Virus and TLR ligands were washed out, cells returned to culture for 24 h prior to collection of cells for measurement of HIV-1 <i>gag</i> DNA by real-time PCR; means and standard deviations are plotted, ** p<0.005 in Student's <i>t</i> test of virus burdens between vehicle and TLR treated cells. N = 4.</p

HIV-1 infection of PBL is resistant to LPS-induced antiviral factor.

<p>Supernatants of MDM activated by LPS were harvested after 4 h and applied to PBL prior to infection by ADA (A) or NL4-3 (B). Infected PBL were cultured for one week prior to collection of supernatants for measurement of p24. N = 2.</p

LPS induces an anti-HIV-1 response in MDM.

<p>MDM from two different donors were treated with vehicle or LPS prior to and during infection with ADA. LPS was washed out with virus (wash) or was replaced for one week culture. Cell supernatants were then harvested for measurement of HIV-1 p24. N (number of experiments with cells from different donors) >4.</p

TBK1 and JAK/STAT are either both required or both dispensable for LPS-induction of previously described antiviral factors.

<p>MDM were activated by LPS in the presence of vehicle, BX-795, or JAK-I and cells were harvested at the indicated times for real-time PCR amplification of the specific transcripts indicated, standardized by GAPDH transcripts as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024193#s4" target="_blank">Materials and Methods</a>, means and standard deviations are plotted. N = 2.</p

LPS activated MDM resist infection by three different strains of HIV-1.

<p>MDM were treated with LPS or vehicle and infected with the virus indicated, LPS was washed out with virus. After one week of culture, cell supernatants were harvested for measurement of HIV-1 p24. N = 2.</p

LPS activated MDM secrete anti-HIV-1 factor (s).

<p>MDM were treated with LPS for 10 min or left untreated, washed, and then supernatants harvested at the times shown. Supernatants were then applied to MDM prior to ADA infection. Infected MDM were cultured four days prior to collection of supernatants for measurement of p24. N>3.</p

Three different TLR ligands induce an anti-HIV-1 response in MDM.

<p>MDM were treated with either vehicle, LPS, R848, or dsRNA at the doses indicated. Cells were treated and infected as described in Fig. 1 with ligands washed out with virus. N = 3.</p

NF-κB is dispensable for the anti-HIV-1 response induced by three different TLR ligands.

<p>(A) MDM were activated and infected by ADA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024193#pone-0024193-g003" target="_blank">Fig 3</a> in the presence of CAPE, PS-1145, or vehicle and were cultured four days prior to collection of supernatants for measurement of p24. N = 2. (B). MDM were treated with the inhibitors shown and LPS and then supernatants were harvested; supernatants were then applied to MDM during ADA infection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024193#s4" target="_blank">Materials and Methods</a>; infected cells were harvested after 24 h for measurement of HIV-1 DNA, means and standard deviations are plotted. N = 3. ** p<0.005 in Student's <i>t</i> test of virus burdens between vehicle and LPS treated cells.</p

IFN-β is dispensable for the anti-HIV-1 response induced by LPS.

<p>(A) MDM were activated by LPS in the presence of vehicle or BX-795, supernatants were collected for the measurement of IFN-β. N = 2. (B) MDM were infected by ADA at the indicated concentrations of JAK-I in the presence of vehicle, supernatants of LPS-activated cells, or recombinant IFN-β. N = 2. (C) MDM were activated by LPS in the presence of vehicle or indicated concentrations of JAK-I and their supernatants were collected and applied to fresh MDM that were infected by ADA. N = 3. All infected cells were harvested after 24 h for measurement of HIV-1 DNA, means and standard deviations are plotted.</p

The combined effects of p38 and JNK MAPK are partially required for the anti-HIV-1 response induced by three different TLR ligands.

<p>Panel A. MDM were activated by LPS and infected by ADA in the presence of SB203580, JNK-I, both inhibitors, or vehicle. N = 3. Panel B. MDM were activated by LPS, R848, or dsRNA and infected by ADA in the presence of SB203580 and JNK-I or vehicle. N = 2. Panel C. MDM were activated by LPS in the presence of vehicle or SB203580 and JNK-I (LPS + SB203580+ JNK-I supt, grey bar). Supernatants were collected and applied to fresh MDM that were infected by ADA in the presence of vehicle or SB203580 and JNK-I (ADA+ SB203580+JNK-I, grey striped bar). N = 3. All infected cells were harvested after 24 h for measurement of HIV-1 DNA, means and standard deviations are plotted. * p<0.05, ** p<0.005, *** p <0.0005 in Student's <i>t</i> test of virus burdens infected cell systems indicated.</p