Pharmacologically-induced cellular stress and herpesvirus replication
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Date
2014-08-14
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Johns Hopkins University
Abstract
Herpesviruses are a family of DNA viruses that infect > 90% of the human population. The three classes of herpesviruses (alpha, beta and gamma) exhibit two viral gene expression programs upon host cell infection. In gammaherpesviruses, latency is characterized by the maintenance of the viral genome in the form of an episome. Viral gene expression is relatively restricted. Lytic gene expression results in in the production of infectious virus and the expression of many more viral proteins. Kaposi sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that plays a role in the pathogenesis of Kaposi sarcoma (KS), multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). Induction of KSHV lytic replication in PEL cell lines is characterized by the sequential expression of immediate early, early and late genes. The replication transactivator protein (RTA) serves as the switch between latent and lytic viral gene expression. Pharmacologic induction of cellular stress has been reported to activate lytic replication in vitro.
In this thesis, data is presented to indicate that ER stress activates lytic gene expression. RT-PCR and immunoblot analysis of KSHV positive PEL cells (BC-3 and BCBL-1) treated with known ER stress inducers such as thapsigargin and tunicamycin led to the induction of the unfolded protein response (UPR) as well as lytic gene expression. We also showed that bortezomib; a proteasome inhibitor, induced ER stress and lytic replication in these cell lines resulting in apoptosis and increased virus production.
Furthermore, nelfinavir, an HIV aspartyl protease, functions as an inhibitor of the production of KSHV in cell lines. Nelfinavir has been reported by others to cause ER stress, autophagy and apoptosis in a variety of cancer cells. We observed the induction of ER stress, autophagy and apoptosis in KSHV cell lines (BC-3 and BCBl-1) treated with nelfinavir by RTPCR, immunoblot analysis and ultrastructural studies. We also showed an increase in the expression of viral lytic genes by RTPCR and immunofluorescence.
Nelfinavir has also been reported to inhibit herpesvirus replication in vitro. In order to determine the stages of replication inhibited by nelfinavir, we studied its effects on transformed and primary cells infected with herpes simplex virus type 1 (HSV-1). We confirmed by plaque reduction and virus titration assays that virus production was indeed inhibited following treatment of infected cells with nelfinavir. Ultrastructural studies showed that DNA replication and packaging of the viral genome into capsids as well as exit from the nucleus occurs in nelfinavir-treated cells. However, viral glycoprotein maturation, secondary envelopment in the cytoplasm and export of mature virus to the extracellular space did not take place. Nelfinavir appears to inhibit late stages of virus replication required for export of infectious virus.
A number of cellular targets of nelfinavir have been identified in a variety of cancer cells. These targets include the 20S proteasome, heat shock protein 90 (hsp90) and the PI3K/AKT pathway. Nelfinavir has also been reported to inhibit a metalloprotease (Site-2-Protease) in liposarcoma. The targets responsible for inhibiting secondary envelopment have not been studied. HIV protease inhibitors have been shown to inhibit FACE-1, a zinc metalloproteases and result in the nuclear membrane dysregulation. A KSHV-positive cell line with a doxycycline inducible FACE-1 shRNA showed an increase in lytic gene expression following treatment with doxycycline. Several HIV protease inhibitors were also studied to determine if they exhibited similar effects on PEL cell lines. When compared to nelfinavir, other HIV protease inhibitors (lopinavir, ritonavir and darunavir) did not induce ER stress or lytic gene expression as robustly. These results suggest that the effects of nelfinavir are unique and require further study. The development of drugs similar to nelfinavir that specifically target the cancer cells, have fewer side effects in patients, and prevent the production and spread of infectious virus may be beneficial for treatment KSHV associated malignancies.
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Keywords
Herpesvirus, Kaposi sarcoma, Nelfinavir, HIV protease inhibitor, Replication