11 research outputs found
Regulation of PI3K/Akt dependent apoptotic markers during b virus infection of human and macaque fibroblasts
<div><p>B virus (<i>Macacine herpesvirus</i> 1), a simplex virus endemic in macaques, causes encephalitis, encephalomyelitis, and death in 80% of untreated zoonotically infected humans with delayed or no treatment. Here we report a significant difference in PI3K/Akt-dependent apoptosis between B virus infected human and macaque dermal fibroblasts. Our data show that B virus infection in either human or macaque fibroblasts results in activation of Akt via PI3K and this activation does not require viral <i>de novo</i> protein synthesis. Inhibition of PI3K with LY294002 results in a significant reduction of viral titers in B virus infected macaque and human fibroblasts with only a modest difference in the reduction of virus titers between the two cell types. We, therefore, tested the hypothesis that B virus results in the phosphorylation of Akt (S473), which prevents apoptosis, enhancing virus replication in B virus infected macaque dermal fibroblasts. We observed markers of intrinsic apoptosis when PI3K activation of Akt was inhibited in B virus infected macaque cells, while, these apoptotic markers were absent in B virus infected human fibroblasts under the same conditions. From these data we suggest that PI3K activates Akt in B virus infected macaque and human fibroblasts, but this enhances virus replication in macaque fibroblast cells by blocking apoptosis.</p></div
Phosphorylation of Akt in B virus infected macaque (RMF) and human cells (HFF).
<p>RMF and HFF cells were exposed either with B virus (MOI 5), mock-infected cell lysate (mock), following treatment with either 50 μM LY294002, DMSO, or medium for 1, 3, and 6 hpi. <b>(A)</b> Cell lysates were collected, fractionated, immunoblotted, and probed for pAkt (S473), using total Akt as a loading control. <b>(B)</b> Virus entry into RMF and HFF cells was synchronized by incubating at 4°C for 1 hour. After 1 hour adsorption at 4°C, cells were transferred to 37°C. Cell lysates were again prepared and probed for pAkt (S473) and total Akt at 0, 5, 15, and 30 minutes at 37°C.These results are representative of 3 independent experiments.</p
PI3K-dependent Akt phosphorylation in B virus infected cells.
<p>RMF and HFF cells were either grown in the presence or absence of 50 μM LY294002 for 2 hours prior to infection and throughout the duration of infection. Cells were collected at 1, 3, 6 hpi, lysed, fractionated, immunoblotted, then probed for pAkt (S473) and total Akt. These results are representative of 3 independent experiments.</p
Effect of PI3K/Akt activation on B virus replication in human and macaque cells.
<p>RMF and HFF cells were grown in the presence or absence of LY294002 for 2 hours prior to infection and the treatment was continued throughout the infection for 24 hours. <b>(A)</b> Cell lysates were collected at 24 hpi and the effect of LY294002 on phosphorylation of Akt (S473) was assessed. <b>(B)</b> B virus titers in infected (MOI 5) RMF/HFF cells with or without LY294002 treatment. <b>(C)</b> B virus infectious titers in infected (MOI 0.2) RMF/HFF cells with or without LY294002 treatment. <b>(D)</b> Relative fold-reduction in infectious virus titers in RMF and HFF cells treated with LY294002. All results are representative of 3 independent experiments.</p
Expression of apoptotic markers in B virus infected macaque and human cells in the absence of PI3K/Akt activation.
<p>RMF and HFF cells were treated in the presence and absence of LY294002 2 hours prior to infection and throughout infection for 24 hours. <b>(A)</b> Cell morphology was observed microscopically. <b>(B)</b> At 24 hpi cells were collected, fractionated, immunoblotted, and probed for cleaved caspase-3, <b>(C)</b> cleaved caspase-7, and <b>(D)</b> cleaved PARP. These results are representative of 3 independent experiments.</p
Phosphorylation of Akt in B virus infected macaque and human cells was independent of <i>de novo</i> protein synthesis.
<p>RMF and HFF cells were grown in the presence or absence of CHX (100 μg/ mL) in serum-free medium 1 hour prior to infection. Cells were infected with B virus (MOI 5) or mock cell lysate diluted in serum-free medium in the presence or absence of CHX. Cell were collected and lysed at 0, 2, 4, and 8 hpi, fractionated, immunoblotted, and probed for pAkt (S473), total Akt, and B virus Us11, the latter to validate CHX inhibition. These results are representative of 3 independent experiments.</p
Overall summary of mAbs discussed in this publication and some of their characteristics.
<p>Overall summary of mAbs discussed in this publication and some of their characteristics.</p
mAbs blocked by pooled macaque and human sera as determined by the mAb-CE.
<p>mAbs blocked by pooled macaque and human sera as determined by the mAb-CE.</p
mAbs competed by human sera as determined by the mAb-CE.
<p>mAbs competed by human sera as determined by the mAb-CE.</p
The relative reactivity of mAbs to viruses of the simplexvirus group was tested by tELISA.
<p>The relative reactivity of mAbs to viruses of the simplexvirus group was tested by tELISA.</p