Since the beginning of the AIDS epidemic, three human herpesviruses have been discovered. DNA fragments of human herpesvirus 8 (HHV-8), also termed Kaposi sarcoma-associated herpesvirus (KSHV) were first identified in 1994 in AIDS associated Kaposi’s sarcoma biopsy specimens by representational difference analysis. DNA of this virus is invariably found in Kaposi’s sarcoma, body cavity based lymphomas, and certain forms of Castleman’s disease.
Entry of herpesviruses into host cells is seen as a multistep process involving several cellular receptors and at least four viral envelope glycoproteins of which only three (glycoproteins H, L and B) are conserved amongst all herpesviruses. The first step is attachment of the virion to the cytoplasma membrane. In most human herpesviruses, this step is mediated by one or more of the virus- or genus-specific glycoprotein(s) which bind to specific cellular receptor(s) and are often responsible for the cell tropism of the respective herpesvirus. In KSHV, glycoprotein gpK8.1 is important for attachment of the virion to the cell by binding to heparansulfate. These strain-specific viral glycoproteins form complexes with the highly conserved glycoproteins H and L (gH/gL), either following receptor binding or already before, and seem to ‘activate’ gH/gL which – at least in some herpesviruses – is followed by endocytotic uptake of the virion. Interaction of activated gH/gL with glycoprotein B (gB) is then required to trigger the last step in herpesvirus entry: Fusion of the virion envelope with cellular membranes. This step is executed by trimeric gB which shares structural similarities with both class I and class II fusion proteins. As described above, a complex formed by the conserved glycoproteins gH/gL and at least one virus-specific receptor-binding protein is required to trigger gB mediated fusion in most herpesviruses.
Although KSHV is able to enter a variety of permanent cell lines, KS pathogenesis is closely linked to infection of endothelial cells (KS) or B-lymphocytes (PEL, MCD). Using immunoprecipitation and mass spectrometry we identified EphA2 as a cellular binding partner of KSHV gH/gL. We showed that EphA2 does not only bind to KSHV gH/gL with high affinity and specificity but that EphA2 is crucial for the infection of endothelial and epithelial cells by KSHV.
As EphA2 is highly expressed in KS lesions, we tested whether latent and/or lytic infection with KSHV enhances EphA2 expression. Surprisingly, whereas EphA2 expression was not altered by latent KSHV infection in epithelial cells, reactivation of the lytic cycle resulted in reduction of EphA2 protein levels by more than 90% (Figure 1). Interestingly, expression of the KSHV lytic-cycle switch RTA was sufficient to downregulate EphA2-expression. RTA harbors domains of a classical transcription factor and upregulates the transcription of several viral and cellular genes. However, it also contains a RING-finger E3-ubiquitin ligase-like domain and has been shown to mediate the downregulation of several host cell proteins. We thus examined whether RTA alters EphA2 expression on transcription and post-transcriptional levels. We were able to show that RTA reduces the amount of EphA2 mRNAs by about 50%. A similar reduction was seen when an EphA2-promoter reporter construct was introduced into RTA-expressing cells. Thus, RTA regulates EphA2 expression both at the level of transcription and by post-transcriptional mechanisms. Surprisingly, inhibition of the proteasome system by the proteasome inhibitor MG132 did not inhibit RTA-mediated downregulation of EphA2.
In order to get a deeper understanding of the EphA2 downregulation mechanism in induced iSLK.RTA and iSLK.219 cells, we wanted to test for (1) the dependence of EphA2 downregulation on the presence of the EphA2 promoter in induced iSLK.RTA and iSLK.219 cells and (2) the dependence of EphA2 downregulation on the presence of the EphA2 intracellular domain in induced iSLK.RTA and iSLK.219 cells. For these purposes, iSLK.RTA and iSLK.219 stable cell lines expressing either a C-terminally FLAG tagged full length EphA2 construct (E2-FLAG) or a FLAG tagged EphA2 construct lacking all intracellular residues (E2ΔIC-FLAG) were established. Induction of RTA expression by treatment with doxycycline resulted in a marked downregulation of EphA2 expression both of the intrinsic protein and of EphA2 expressed from a retroviral construct. The intracellular domain was not required for this effect (Figure 2).