Ets-1-dependent expression of vascular endothelial growth factor receptors is activated by latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus through interaction with Daxx

Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in Kaposi's sarcoma (KS) lesion and play a key role in angiogenesis. Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) has multiple functions related to viral latency and KSHV-induced oncogenesis. In this report, we have identified Daxx as a LANA-binding protein by co-immunoprecipitation analysis of HeLa cells stably expressing LANA. LANA associated with Daxx in a PEL cell line infected with KSHV. LANA and Daxx also bound in vitro, suggesting direct interaction. From the results of binding assays, a region containing the Glu/Asp-rich domain within LANA, and a central region including the second paired amphipathic helix within Daxx contributed to the interaction. To address the physiological significance of this interaction, we focused on a Daxx-mediated VEGF receptor gene regulation. We found that Daxx repressed Ets-1-dependent Flt-1/VEGF receptor-1 gene expression, and that LANA inhibited the repression by Daxx in a reporter assay. Analyses of flow cytometry and real-time PCR revealed that expression of VEGF receptor-1 and -2 in LANA-expressing human umbilical vein endothelial cells (HUVECs) significantly increased. Co-immunoprecipitation and immunoblotting experiments suggested that LANA-bound Daxx to inhibit the interaction between Daxx and Ets-1. Chromatin immunoprecipitation assays showed that Daxx associated with VEGF receptor-1 promoter in HUVECs, and that LANA expression reduced this association. These results suggested that LANA contributes to a high expression of VEGF receptors in KS lesion by interfering with the interaction between Daxx and Ets-1.     

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 RTA reactivates murine gammaherpesvirus 68 from latency

Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.     

Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

The availability of reliable recombinant reporter virus systems has been a great boon to the study of Kaposi''s sarcoma-associated herpesvirus (KSHV/HHV-8). Unexpectedly, we found that expression of the ostensibly constitutive green fluorescent protein (GFP) marker was progressively lost during unselected passage in primary rat mesenchymal precursor cells (MM), despite efficient maintenance of latent viral gene expression and episomal partitioning. This repression of EF1-α promoter-driven GFP expression appeared to be passage-dependent, however, since functionally immortalized MM cells derived from long serial passage retained stable expression of GFP following rKSHV.219 infection. Chromatin analysis of cultures that we had infected in parallel demonstrated an increase in repressive H3K27 tri-methylation across the viral episome with the exception of the LANA control region in MM cells infected at early rather than late passage post-isolation. The silencing of GFP expression in the MM cells was reversible in a dose-dependent fashion by the histone deacetylase inhibitor valproic acid, further implicating cellular silencing on incoming viral genomes, and underscoring potential differences in viral gene regulation between primary and functionally immortalized cells. Furthermore, using multispectral imaging flow cytometry, we also determined that the extent of GFP expression per cell among those that were positive did not correlate with the number of LANA dots per nucleus nor the extent of overall LANA expression per cell. This suggests a more complex mode of local gene regulation, rather than one that simply reflects the relative intracellular viral copy number. In sum, we have demonstrated the significant potential for false-negative data when using a constitutive marker gene as a sole means of evaluating herpesviral infection, especially in primary cells.