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 LCV EBNA1 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.

FIG. 1

Amino acid alignment of rhesus LCV, baboon LCV, and EBV EBNA1. Similar or identical residues in all three proteins are indicated by an asterisk and in two proteins by a period. GAR motifs in rhesus and baboon LCVs are underlined. BamHI and NcoI restriction enzyme sites used for C-terminal epitope insertion and deletion of the GAR in rhesus EBNA1 are highlighted.

FIG. 2

Rhesus LCV EBNA1 supports ori-p-dependent plasmid maintenance and replication. (A) Frequency of hygromycin-resistant growth after cotransfection of hygromycin phosphotransferase containing BSAII plasmid with EBV EBNA1 (■), rhesus LCV EBNA1 (▴), or vector control (○) is shown. (B) Replication of ori-p plasmid DNA in eukaryotic cells is shown as the copy number of DpnI-resistant DNA in Hirt extracts from BJAB cells cotransfected with an ori-p plasmid and vector control, a construct with EBV EBNA1 in the antisense (AS) orientation, and EBV EBNA1, baboon LCV EBNA1, and rhesus LCV EBNA1 expression constructs. Results from two representative experiments are shown as the mean and the standard deviation of duplicate PCR measurements.

FIG. 3

(A) Schematic representation of EBV EBNA1, EBV EBNA1-deleted for the GAR domain (E1dGAR), and the EBV EBNA1/rhesus or baboon GAR chimeras (E1rhGAR and E1baGAR). The BspEI site within EBV EBNA1 used for insertion of the rhesus and baboon GARs is shown, as is the location of the B*3501-restricted epitope (HPVGEADYFEY) and the A*0203-restricted epitope (VLKDAIKDL) within EBV EBNA1. (B) Western blot of recombinant vaccinia viruses expressing EBV EBNA1 (vacc [EBNA1]) and chimeric EBNA1s containing the rhesus LCV EBNA1 GAR (vaccE1rhGAR), baboon LCV GAR (vaccE1baGAR), or no GAR (vaccE1dGAR). BS-C-1 cells were infected with recombinant vaccinia viruses for 15 h, and extracts of 2 × 10⁵ cell equivalents were then probed with the EBNA1-specific monoclonal antibody 1H4-1. EBV EBNA1 expression is driven by a T7 promoter and requires coinfection with a T7-expressing vaccinia virus (vaccT7). vaccNB55 and vaccNB43 are control viruses with E1rhGAR and E1baGAR cloned in the reverse orientation.

FIG. 4

CTL recognition of EBV EBNA1/rhesus GAR and EBV EBNA1/baboon GAR chimeric proteins endogenously expressed in a human B-cell background. (A) HLA B*3501-positive LCL infected with recombinant vaccinia viruses expressing EBNA1/T7 (▸), EBNA1 deleted of the GAR domain (●), EBNA1/rhGAR (▴), EBNA1/baGAR (■), or EBNA3A (◊) were used as targets for EBV EBNA1 epitope 407-417-specific B35-restricted CTL effectors. (B) HLA A*0203-positive LCL infected with recombinant vaccinia viruses as in panel A were used as targets for EBNA1-specific A*0203-restricted EBV EBNA1 epitope 574-582 CTL effectors. For each graph, LCL pulsed with the cognate epitope peptide (▾) or DMSO (○) are shown as controls. Results are displayed as the percent specific lysis at the indicated effector/target ratio (E:T).

FIG. 5

CTL recognition of EBV EBNA1/rhesus GAR and EBV EBNA1/baboon GAR chimeric proteins endogenously expressed in a simian B-cell background. LCL of rhesus (A) or baboon (B) origin were used as target cells in CTL assays with EBV EBNA1 epitope 407-417-specific B35-restricted CTL effectors. The human HLA B*3501 allele was provided by coinfection with a recombinant vaccinia virus expressing the HLA B35 heavy chain. Target cells were infected with the recombinant vaccinia virus vaccB*3501 (□), or coinfected with vaccB*3501 and vaccE1dGAR (●), vaccE1rhGAR (▴), or vaccE1baGAR (■), and vaccEBNA1/vaccT7 (▸). For controls, simian LCLs were infected with vaccB*3501 and pulsed with the EBV EBNA1 epitope 407-417 peptide (HPVGEADYFEY) (▾) or DMSO (○). Results are displayed as the percent specific lysis at the indicated effector/target ratio (E:T).

FIG. 6

Construction of rhesus LCV EBNA1 expression vectors containing an SIV-gag epitope and presentation of the gag epitope to SIV-gag-specific CTL. (A) Schematic diagrams of the rhesus LCV EBNA1 chimeras are shown at the top highlighting the relative positions of the flag epitope (flg), SIV-gag epitope (gag) and GAR domain. Expression of the rhesus LCV EBNA1 chimeras by using recombinant adenoviruses is shown in the Western blot with a flag-specific monoclonal antibody. 293 cells were infected with the indicated recombinant adenovirus; 48 h later cells were harvested, and extracts were probed with a FLAG-specific antibody. (B and C) SIV-gag-specific CTL activity against Mamu A*01 fibroblast targets infected with recombinant adenoviruses expressing rhesus LCV EBNA1 chimeras or B-cell targets infected with recombinant vaccinia virus expressing wild-type SIV-gag is shown. Constructs containing the SIV-gag epitope are shown as solid symbols and constructs without the SIV-gag epitope are shown as open symbols. Results are displayed as the percent specific lysis at the indicated effector/target ratio (E:T).