Subversion of Natural Killer Cell Defenses Induced by a Deadly Zoonotic Virus

B virus (Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpes B virus) is an Old World monkey simplex virus endemic in macaques. B virus infection in its natural host, macaque, is very similar to HSV-­‐1 infection in humans causing mild or asymptomatic infection. On the other hand, zoonotic infection in humans results in death in the absence of early initiation of antiviral drugs. Viruses evade host immune responses in order to survive and propagate. Most herpes viruses including HSV-­‐1 down-­‐regulate major histocompatibility complex class I (MHC class I) surface expression on infected cells in order to prevent CD8+ T-­‐cell recognition and subsequent cell lysis. MHC class I molecules bind to the inhibitory receptors of NK cells and prevent NK cell activity. Thus, this mechanism protects HSV-­‐1 infected cells from CD8+ T-­‐cell lysis, making them sensitive to natural killer (NK) cell cytotoxicity. To investigate if B virus pathogenicity is a result of novel immune evasion mechanisms employed by B virus, we determined NK cell regulation during B virus infection. To this end, our experiments demonstrate that B virus does not down-­‐ regulate MHC I expression as effectively as HSV-­‐1, leading us to hypothesize that B virus in-­‐ fected cells are resistant to NK cell activity. We examined the expression of MHC I chain related genes (MICA/ MICB), which are activation ligands to NKG2D receptors on NK cells. Our results show that there is no significant difference in MICA and MICB expression between HSV-­‐1 and B virus infected cells. Furthermore, we tested for the up-­‐regulation of cytokines and chemokines responsible for NK cell activation and migration. Our results indicate a significant up-­‐regulation of IFN-­‐α from PBMCs co-­‐cultured with HSV-­‐1 infected cells, which plays an important role in activating NK cells. NK cells within these PBMCs up-­‐regulate perforin release indicative of NK cell activity. PBMCs co-­‐cultured with B virus infected cells do not up-­‐regulate any cytokines or chemokines responsible for NK cell activity. As a result the NK cells within these PBMCs do not significantly up-­‐regulate perforin release. These results demonstrate that B virus employs a novel immune evasion mechanism to subvert NK cell activity

PNI Biomarkers and Health Outcomes in College Women

Sleep disturbance has been found to trigger a stress response with a subsequent activation of the psychoneuroimmunological (PNI) pathway associated with adverse health outcomes. This study aimed to assess the association among selected PNI biomarkers, sleep disturbances, and adverse health outcomes (depressive symptoms, physical symptoms). A stratified, quota sample (14 poor sleepers and 15 good sleepers) was drawn from a pool of healthy college women from a larger scale of study. The participants reported their sleep, stress, depressive, and physical symptoms. Wrist actigraphy was used to collect objective sleep data, and the Enzyme-Linked ImmunoSorbent Assay was used to assess PNI biomarkers. Poor sleep quality, higher stress perception, elevated serum serotonin, and lower serum interleukin-10 explained 75.3% of the variances for the depressive symptoms. Poor sleep quality along with delayed peak activity rhythms accounted 31.4% of the physical symptoms. High serotonin and tumor necrosis factor-α were the significant predictors for poor sleep efficiency, and serotonin was the single significant predictor for poor daytime functioning. Stress and sleep disturbances negatively impact the health of college women and should be as part of regular check-ups on campus. PNI effects on health outcomes should be further explored. Educational materials in the areas of sleep hygiene, health impacts from sleep disturbances, and strategies to maintain synchronized circadian rhythms should be mandatorily included in the college curriculum

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

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

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