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Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

ABSTRACT: Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells.

SUBMITTER: Yamamoto T

PROVIDER: S-EPMC4789179 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

Yamamoto Takenobu T Ali Mir A MA Liu XueQiao X Cohen Jeffrey I JI

Virology 20140129

Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but n ...[more]

PMID: 24606682

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Project description:Immune regulation of alphaherpesvirus latency and reactivation is critical for the control of virus pathogenesis. This is evident for herpes simplex virus 1 (HSV-1), where cytotoxic T lymphocytes (CTLs) prevent viral reactivation independent of apoptosis induction. This inhibition of HSV-1 reactivation has been attributed to granzyme B cleavage of HSV infected cell protein 4 (ICP4); however, it is unknown whether granzyme B cleavage of ICP4 can directly protect cells from CTL cytotoxicity. Varicella zoster virus (VZV) is closely related to HSV-1; however, it is unknown whether VZV proteins contain granzyme B cleavage sites. Natural killer (NK) cells play a central role in VZV and HSV-1 pathogenesis and, like CTLs, utilize granzyme B to kill virally infected target cells. However, whether alphaherpesvirus granzyme B cleavage sites could modulate NK cell-mediated cytotoxicity has yet to be established. This study aimed to identify novel HSV-1 and VZV gene products with granzyme B cleavage sites and assess whether they could protect cells from NK cell-mediated cytotoxicity. We have demonstrated that HSV ICP27, VZV open reading frame 62 (ORF62), and VZV ORF4 are cleaved by granzyme B. However, in an NK cell cytotoxicity assay, only VZV ORF4 conferred protection from NK cell-mediated cytotoxicity. The granzyme B cleavage site in ORF4 was identified via site-directed mutagenesis and, surprisingly, the mutation of this cleavage site did not alter the ability of ORF4 to modulate NK cell cytotoxicity, suggesting that ORF4 has a novel immunoevasive function that is independent from the granzyme B cleavage site.IMPORTANCE HSV-1 causes oral and genital herpes and establishes life-long latency in sensory ganglia. HSV-1 reactivates multiple times in a person's life and can cause life-threatening disease in immunocompromised patients. VZV is closely related to HSV-1, causes chickenpox during primary infection, and establishes life-long latency in ganglia, from where it can reactivate to cause herpes zoster (shingles). Unlike HSV-1, VZV only infects humans, and there are limited model systems; thus, little is known concerning how VZV maintains latency and why VZV reactivates. Through studying the link between immune cell cytotoxic functions, granzyme B, and viral gene products, an increased understanding of viral pathogenesis will be achieved.

Project description:Varicella and herpes zoster are mild symptoms-associated diseases caused by varicella-zoster virus (VZV). They often cause severe complications (disseminated zoster), leading to death when diagnoses and treatment are delayed. However, most commercial VZV diagnostic tests have low sensitivity, and the most sensitive tests are unevenly available worldwide. Here, we developed and validated a highly sensitive VZV diagnostic kit based on the chemiluminescent immunoassay (CLIA) approach. VZV-glycoprotein E (gE) was used to develop a CLIA diagnostic approach for detecting VZV-specific IgA, IgG, and IgM. The kit was tested with 62 blood samples from 29 VZV-patients classified by standard ELISA into true-positive and equivocal groups and 453 blood samples from VZV-negative individuals. The diagnostic accuracy of the CLIA kit was evaluated by receiver-operating characteristic (ROC) analysis. The relationships of immunoglobulin-isotype levels between the two groups and with patient age ranges were analyzed. Overall, the developed CLIA-based diagnostic kit demonstrated the detection of VZV-specific immunoglobulin titers depending on sample dilution. From the ELISA-based true-positive patient samples, the diagnostic approach showed sensitivities of 95.2%, 95.2%, and 97.6% and specificities of 98.0%, 100%, and 98.9% for the detection of VZV-gE-specific IgA, IgG, and IgM, respectively. Combining IgM to IgG and IgA detection improved diagnostic accuracy. Comparative analyses on diagnosing patients with equivocal results displaying very low immunoglobulin titers revealed that the CLIA-based diagnostic approach is overall more sensitive than ELISA. In the presence of typical VZV symptoms, CLIA-based detection of high titer of IgM and low titer of IgA/IgG suggested the equivocal patients experienced primary VZV infection. Furthermore, while no difference in IgA/IgG level was found regarding patient age, IgM level was significantly higher in young adults. The CLIA approach-based detection kit for diagnosing VZV-gE-specific IgA, IgG, and IgM is simple, suitable for high-throughput routine analysis situations, and provides enhanced specificity compared to ELISA.

Dataset Information

Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

Publications

Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

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