Background

The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.

Methodology/Principle Findings

Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.

Conclusions/Significance

We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r-/- or ebi-3-/- mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r-/- or ebi-3-/- mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r-/- or ebi-3-/- mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines.

Subfertility is a major concern of long-term cancer survivors at the reproductive age. We have previously demonstrated that a potent humanin analogue, HNG, protected chemotherapy-induced apoptosis in germ cells but not cancer cells in a metastatic melanoma allograft model. In this study, we utilized severe combined immuno-deficiency (SCID) mice bearing human medulloblastoma to study the effect of HNG in Temozolomide (TMZ) induced male germ cell apoptosis and white blood cell (WBC) suppression. Human medulloblastoma DAOY cells were injected subcutaneously into the right flank of male SCID mice. Three weeks later, groups of tumor-bearing mice received one of the following treatments: vehicle, HNG, TMZ, or TMZ + HNG. 24 h after last injection, the tumors weights, complete blood counts, liver and spleen weights, male germ cell apoptosis was assessed. HNG did not affect TMZ's significant anti-tumor action. HNG significantly prevented TMZ-induced germ cell apoptosis and attenuated the suppressed total WBC and granulocyte counts in SCID mice with or without TMZ treatment. HNG also attenuated TMZ-induced body weight loss and decrease of spleen and liver weights. In conclusion, HNG ameliorated TMZ-induced germ cell apoptosis; WBC and granulocytes loss; and decreased body/organ weights without compromising the TMZ's anti-cancer action on medulloblastoma xenografts in SCID mice.