Reactivation of Kaposi's Sarcoma-Associated Herpesvirus Infection from Latency by Expression of the ORF 50 Transactivator, a Homolog of the EBV R Protein

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is a lymphotropic virus strongly linked to several AIDS-related neoplasms. The primary reservoir of infection consists of latently infected B lymphocytes and possibly other mononuclear cells. Viral reactivation from latency and spread from this lymphoid reservoir is presumably required for development of nonlymphoid tumors like KS. Here we show that deregulated expression of a single viral gene, ORF 50, which encodes a transactivator able to selectively upregulate delayed-early viral genes, suffices to disrupt latency and induce the lytic gene cascade in latently infected B cells. The identification of this gene opens the way to studies of the physiologic mechanisms controlling reactvation of KSHV from latency.

The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jκ (CSL), the target of the Notch signaling pathway

“…The full-length RTA (1-691 aa) or 50⌬STAD (1-530 aa) coding sequence, obtained by PCR using 5Ј primer (5Ј-GCGCGAA TTCATGGCGCAAGATGACAAGGGTAAG-3Ј) and 3Ј primer (5Ј-GCGCGTCGACTCAGTCTCGGAAGTAATTACGCCA-3Ј or 5Ј-GCGCGTCGACTCACTTACGCTTCTTTGAGCTCCTC TC-3Ј) from template pcDNA3-Flc50 (Lukac et al 1998), was inserted into pBD-Gal4-Cam (Clontech) at the EcoRI and XhoI sites. These two plasmids, pBD-Gal4-50 and pBD-Gal4-50⌬STAD, were used in yeast two-hybrid screening as baits.…”

“…Uninduced or TPA-induced BCBL-1 cells were lysed with RIPA buffer. Immunoprecipitation and subsequent Western blot were essentially the same as described above, except for using rabbit anti-RBPJ polyclonal antibody (Cemine) in immunoprecipitation and rabbit anti-ORF50 antiserum (Lukac et al 1998) in Western blot. As a control for endogenous coimmunoprecipitation, OT11 (RBPJ −/− ) cells were transfected with pcDNA3-Flc50.…”

“…Ectopic expression of RTA in latently infected B cells triggers efficient lytic reactivation (Lukac et al 1998;Sun et al 1998;Gradoville et al 2000), and spontaneous reactivation is blocked by dominant-negative mutants of RTA (Lukac et al 1999). RTA is the homolog of the EBV BRLF1 gene product, a sequence-specific DNA-binding protein known to function as a transcriptional activator (Quinlivan et al 1993;Russo et al 1996;Zalani et al 1996;Ragoczy et al 1998).…”

“…RTA is the homolog of the EBV BRLF1 gene product, a sequence-specific DNA-binding protein known to function as a transcriptional activator (Quinlivan et al 1993;Russo et al 1996;Zalani et al 1996;Ragoczy et al 1998). Not surprisingly, KSHV RTA also acts to promote transcriptional activation of a variety of viral promoters, including those for delayed-early viral genes like TK, SSB, the posttranscriptional activator MTA, and the noncoding polyadenylated nuclear RNA, PAN (Lukac et al 1998). KSHV RTA contains an N-terminal DNA-binding domain and a C-terminal activation domain (Lukac et al 1998(Lukac et al , 2001Seaman et al 1999), and direct, high-affinity binding of purified, recombinant RTA to a site in the PAN promoter has been reported (Song et al 2001).…”

“…Not surprisingly, KSHV RTA also acts to promote transcriptional activation of a variety of viral promoters, including those for delayed-early viral genes like TK, SSB, the posttranscriptional activator MTA, and the noncoding polyadenylated nuclear RNA, PAN (Lukac et al 1998). KSHV RTA contains an N-terminal DNA-binding domain and a C-terminal activation domain (Lukac et al 1998(Lukac et al , 2001Seaman et al 1999), and direct, high-affinity binding of purified, recombinant RTA to a site in the PAN promoter has been reported (Song et al 2001). However, other sites that are activated by RTA in vivo-for example, those in the MTA and RAP (K8, K-bZIP) promoters-display no homology to the PAN promoter site, raising the question of whether RTA might have other modes of DNA interaction, such as binding to cellular DNA binding factors.…”