Inhibition of Antigen Presentation by the Glycine/Alanine Repeat Domain Is Not Conserved in Simian Homologues of Epstein-Barr Virus Nuclear Antigen 1

Abstract

Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCVEBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

Translation efficiency of EBNA1 encoded by lymphocryptoviruses influences endogenous presentation of CD8+ T cell epitopes

“…Although one may argue that the Rh-EBNA1 protein is differentially processed in human and primate cells, data presented in this study clearly show that both human and simian cells efficiently process CTL epitopes from Rh-EBNA1. These observations are also supported by earlier studies of Blake and colleagues, who showed that rhesus B cells and fibroblasts expressing Rh-EBNA1 containing a model CTL epitope from the simian immunodeficient virus are efficiently recognized by virus-specific CTL [13]. Furthermore, the impairment of translation and endogenous processing of Rh-EBNA1 following substitution of its native GAr domain with GAr sequences from Hu-EBNA1 highlights the impact of varying GAr sequences on the self-synthesis of EBNA1 homologues.…”

“…However, extensive analysis of rhesus LCV EBNA1-specific CTL expanded from LCV-infected animals revealed that these effector cells fail to recognize full-length EBNA1 protein, while a sequence in which internal repeats were deleted was efficiently recognized [12]. These observations contradict the previous studies by Blake and colleagues who showed that in contrast to EBV EBNA1, the internal repeat sequences within rhesus EBNA1 do not inhibit endogenous processing of CTL epitopes from this protein [13]. These contrasting observations raise important questions regarding the overall biology of LCV infection and the mechanism by which the strongly dominant EBNA1-specific CD8 + T cell responses can be generated in LCV-infected animals.…”

“…We hypothesized that the EBNA1 sequence encoded by simian LCV may have evolved in such a way that it allows more efficient presentation to the CD8 + T cells. Indeed, this hypothesis was to some degree supported by earlier studies published by Blake and colleagues, who showed that CTL epitopes within the EBNA1 sequence from rhesus and baboon EBNA1 are more efficiently processed and presented when compared to EBNA1 derived from EBV [13]. However, these studies provided no mechanistic explanation for the observations.…”

“…Full-length EBV-encoded EBNA1 (referred to as Hu-EBNA1) and GAr-deleted EBNA1 (referred to as Hu-EBNA1DGA) were cloned into the expression vector pcDNA3.1 (Invitrogen) as described previously [28]. In addition, LCV EBNA1 sequences from rhesus (referred to as Rh-EBNA1) and baboon (referred to as Ba-EBNA1) were amplified from the plasmids pSG5pst22c4 and pSG5HPE1c33 [13] , respectively, by PCR and cloned into the expression vector pcDNA3.1. Hu-EBNA1, Hu-EBNA1DGA, Rh-EBNA1, Rh-EBNA1DGA, Ba-EBNA1 and Ba-EBNA1DGA sequences were also sub-cloned in-frame with a sequence coding for green fluorescent protein (pEGFP-N1, Clontech, Palo Alto, CA) to generate Hu-EBNA1-GFP, Hu-EBNA1DGA-GFP, Rh-EBNA1-GFP, Rh-EBNA1DGA-GFP, Ba-EBNA1-GFP and Ba-EBNA1DGA-GFP.…”

Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits

“…Vaccinia viruses expressing HLA alleles B*3501 and B*2704 have been described previously. 20,21 Recombinant viruses expressing A*2501 and B*4402 were constructed using standard procedures. 22 Vaccinia infections were performed at a multiplicity of infection of 10 for 1 hr at 37u.…”