Abstract
Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to therapeutic development through the use of anti-sense strategies or small molecules targeting EBNA1 mRNA structure.
Highlights
Members of the viral family Herpesviridae, which are widely distributed throughout the animal kingdom, are characterized by their large double-stranded, linear DNA genomes
We demonstrate that altered peptide sequences resulting from frameshift mutations within the repeat do not alleviate the immune-evasive function of EBV nuclear protein 1 (EBNA1), suggesting that the repetitive purine-rich mRNA sequence itself is responsible for inhibiting EBNA1 synthesis and subsequent poor immunogenicity
Our comparative analysis of the mRNA sequences of the corresponding repeat regions of different gammaherpesvirus maintenance homologues to EBNA1 highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences
Summary
Members of the viral family Herpesviridae, which are widely distributed throughout the animal kingdom, are characterized by their large double-stranded, linear DNA genomes. The gammaherpesviruses, one of three sub-families of Herpesviridae, predominantly replicate and persist in lymphoid cells with the distinguishing characteristic that they are able to establish lifelong latent infections of their hosts [1]. Latent infection of host cells by many gammaherpesviruses is dependent upon the expression of a viral maintenance protein, which ensures persistence of the viral episome within actively dividing cells, yet simultaneously evades immune surveillance [5,6,7,8]. Expression of the EBV nuclear protein 1 (EBNA1) is widespread in all forms of EBV infection, accentuating its central role in the maintenance of the viral DNA episome, a process essential for viral persistence and associated oncogenic potential [17,18]. More recent studies including reports from the Hoeben group, have proposed that the EBNA1 purine-rich mRNA secondary structure encoding the GAr, rather than the protein sequence, is the critical component
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