scholarly journals Cellular Stress Response to Varicella-Zoster Virus Infection of Human Skin Includes Highly Elevated Interleukin-6 Expression

2018 ◽  
Vol 5 (6) ◽  
Author(s):  
Keith W Jarosinski ◽  
John E Carpenter ◽  
Erin M Buckingham ◽  
Wallen Jackson ◽  
Kevin Knudtson ◽  
...  
Keyword(s):  
Stress Response ◽  
Varicella Zoster Virus ◽  
Human Skin ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Varicella Zoster Virus Infection ◽  
Varicella Zoster ◽  
Protein Levels ◽  
Skin Explants ◽  
Infected Skin

Abstract Background The infectious cycle of varicella-zoster virus (VZV) after reactivation from the dorsal root ganglia includes replication and assembly of complete enveloped virions in the human skin to cause the characteristic herpes zoster (shingles). Methods To pursue studies of innate immunity to VZV infection, we have adapted a fetal skin organ culture model to a human neonatal foreskin explant model. Results Abundant expression of VZV IE62, gE, and gC was visualized by confocal microscopy while numerous enveloped virions were observed by electron microscopy in infected skin organ cultures. Microarray experiments demonstrated that the patterns of upregulated transcripts differed between VZV-infected cells and VZV-infected skin explants. One result stood out, namely a >30-fold elevated interleukin (IL)-6 level in the infected skin explant that was not present in the infected monolayer culture. The IL-6 results in the polyermase chain reaction (PCR) assay were reproduced by quantitative PCR testing with newly designed primers. To determine if increased transcription was accompanied by increased IL-6 expression, we quantitated the levels of IL-6 protein in the explant media at increasing intervals after infection. We found a statistically significant increase in IL-6 protein levels secreted into the media from VZV-infected skin explants as compared with mock-infected explants. Conclusions The cellular stress response to VZV infection in neonatal skin explants included highly elevated levels of IL-6 transcription and expression. This skin organ model could be adapted to other viruses with a skin tropism, such as herpes simplex virus.

scholarly journals Loss of FVIII Expression by Possible Interlinked Immune and Cellular Stress Response in Hemophilia A Mice Treated with AAV Gene Therapy

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Christopher Rogers ◽  
Thais Bertolini ◽  
Roland Herzog
Keyword(s):  
Gene Therapy ◽  
Stress Response ◽  
Hemophilia A ◽  
Genetic Disorder ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Gradual Decline ◽  
Protein Levels ◽  
Time Treatment ◽  
Copy Numbers

Background  Hemophilia A is an X-linked genetic disorder caused by a mutation in the gene for factor VIII (FVIII) protein that reduces the ability of blood to clot. Clinical drug trials have shown the potential of adeno-associated virus (AAV) gene therapy as a one-time treatment for hemophilia A that can produce sustained high levels of FVIII. However, a gradual decline in protein levels has been observed in patients after 2-4 years. The hypothesis being tested in the Herzog Lab is that an interlinked immune and cellular stress response could be causing the loss of expression.     Methods  Two groups of Hemophilia A mice were administered AAV therapy, with one group receiving recurrent doses of Rapamycin. Blood samples were taken at weeks 4, 8, 12 and 14. Mice were euthanized at weeks 4, 8, and 14, and their livers were harvested. qPCR was used to measure AAV copy numbers and FVIII mRNA at 4, 8, and 14 weeks. Cryosections of mice livers from weeks 4, 8, and 14 were stained with antibodies for FVIII protein and CD8.    Results  qPCR showed roughly half as much AAV copy numbers in the rapamycin group at all time points, and little difference in FVIII mRNA between the groups. There was also a large decrease in AAV copy numbers and FVIII mRNA in both groups between 8 and 14 weeks. Immunohistochemistry showed less CD8 and more FVIII signal in mice treated with rapamycin.    Discussion  Experiments are currently being performed to investigate the decline in AAV copy numbers and mRNA between weeks 8 and 14. The immunohistochemistry data shows a relationship between increased FVIII protein levels and decreased cellular immune response but does not explain the gradual decline in FVIII. Further investigation into FVIII expression following AAV gene therapy could lead to an effective one-time treatment for hemophilia A.   

scholarly journals Varicella-Zoster Virus Infection of Human Foreskin Fibroblast Cells Results in Atypical Cyclin Expression and Cyclin-Dependent Kinase Activity

2006 ◽  
Vol 80 (11) ◽  
pp. 5577-5587 ◽  
Author(s):  
Stacey A. Leisenfelder ◽  
Jennifer F. Moffat
Keyword(s):  
Cell Cycle ◽  
Varicella Zoster Virus ◽  
Cultured Cells ◽  
Cyclin B1 ◽  
Human Infection ◽  
Dermal Fibroblasts ◽  
Cyclin D3 ◽  
Varicella Zoster Virus Infection ◽  
Varicella Zoster ◽  
Protein Levels

ABSTRACT In its course of human infection, varicella-zoster virus (VZV) infects rarely dividing cells such as dermal fibroblasts, differentiated keratinocytes, mature T cells, and neurons, none of which are actively synthesizing DNA; however, VZV is able to productively infect them and use their machinery to replicate the viral genome. We hypothesized that VZV alters the intracellular environment to favor viral replication by dysregulating cell cycle proteins and kinases. Cyclin-dependent kinases (CDKs) and cyclins displayed a highly unusual profile in VZV-infected confluent fibroblasts: total amounts of CDK1, CDK2, cyclin B1, cyclin D3, and cyclin A protein increased, and kinase activities of CDK2, CDK4, and cyclin B1 were strongly and simultaneously induced. Cyclins B1 and D3 increased as early as 24 h after infection, concurrent with VZV protein synthesis. Confocal microscopy indicated that cyclin D3 overexpression was limited to areas of IE62 production, whereas cyclin B1 expression was irregular across the VZV plaque. Downstream substrates of CDKs, including pRb, p107, and GM130, did not show phosphorylation by immunoblotting, and p21 and p27 protein levels were increased following infection. Finally, although the complement of cyclin expression and high CDK activity indicated a progression through the S and G2 phases of the cell cycle, DNA staining and flow cytometry indicated a possible G1/S blockade in infected cells. These data support earlier studies showing that pharmacological CDK inhibitors can inhibit VZV replication in cultured cells.

scholarly journals Varicella-Zoster Virus Infection Induces Autophagy in both Cultured Cells and Human Skin Vesicles

2009 ◽  
Vol 83 (11) ◽  
pp. 5466-5476 ◽  
Author(s):  
Marie-Noëlle Takahashi ◽  
Wallen Jackson ◽  
Donna T. Laird ◽  
Timothy D. Culp ◽  
Charles Grose ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Human Skin ◽  
Cultured Cells ◽  
Normal Skin ◽  
Viral Gene ◽  
Varicella Zoster Virus Infection ◽  
Specific Cell ◽  
Late Time ◽  
Specific Staining ◽  
Varicella Zoster

ABSTRACT When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.

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