Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I-restricted cryptic epitope expression

Abstract

Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.

Figure 1

Presentation of an epitope in a downstream alternative reading frame depends upon the context and number of upstream initiation codons in the primary reading frame. (a) MHC class I presentation cassette. Two MHC class I–restricted epitopes, NP_50–57 and NP_147–155, were placed in alternate RFs, each translated by different AUG initiation codons. The first AUG encountered by the scanning ribosome is in variable context and initiates translation of NP_147–155. The second AUG is in excellent context and initiates translation of NP_50–57. After NP_147–155, the sequence of NP is unaltered. Start codons or their null equivalents are indicated in bold. The −3 and +4 nucleotides involved in changing the initiation potential are underlined. (b) Scanthrough is an efficient mechanism of translation from alternative RFs. The MHC class I presentation cassette variants were recombined into the vac genome and tested for sensitization of L-K^d cells for lysis in a standard ⁵¹Cr release assay. Specificity of the T_CD8+ effector population, generated as described in Materials and Methods, is indicated above each data set. The effector/target ratios shown are 30:1 for recognition of NP_147–155 and 13:1 for NP_50–57, respectively. Similar results were observed with three other ratios.

Figure 1

Presentation of an epitope in a downstream alternative reading frame depends upon the context and number of upstream initiation codons in the primary reading frame. (a) MHC class I presentation cassette. Two MHC class I–restricted epitopes, NP_50–57 and NP_147–155, were placed in alternate RFs, each translated by different AUG initiation codons. The first AUG encountered by the scanning ribosome is in variable context and initiates translation of NP_147–155. The second AUG is in excellent context and initiates translation of NP_50–57. After NP_147–155, the sequence of NP is unaltered. Start codons or their null equivalents are indicated in bold. The −3 and +4 nucleotides involved in changing the initiation potential are underlined. (b) Scanthrough is an efficient mechanism of translation from alternative RFs. The MHC class I presentation cassette variants were recombined into the vac genome and tested for sensitization of L-K^d cells for lysis in a standard ⁵¹Cr release assay. Specificity of the T_CD8+ effector population, generated as described in Materials and Methods, is indicated above each data set. The effector/target ratios shown are 30:1 for recognition of NP_147–155 and 13:1 for NP_50–57, respectively. Similar results were observed with three other ratios.

Figure 2

Presentation of NP_50–57 from selected constructs. The indicated constructs were tested for target cell sensitization in a standard ⁵¹Cr release assay.

Figure 3

Reversal of the cassette. (a) Two base insertions, indicated by underline, were made to the original cassette resulting in pairing of RF0 ATG with NP_50–57 and the RF-1 ATG with NP_147–155. (b) Scanning beyond an AUG in excellent context is not sufficient to allow presentation of NP_147–155. The indicated reverse and control constructs were tested for target cell sensitization in a standard ⁵¹Cr release assay. Specificity of the effector populations is indicated above each data set.

Figure 3

Reversal of the cassette. (a) Two base insertions, indicated by underline, were made to the original cassette resulting in pairing of RF0 ATG with NP_50–57 and the RF-1 ATG with NP_147–155. (b) Scanning beyond an AUG in excellent context is not sufficient to allow presentation of NP_147–155. The indicated reverse and control constructs were tested for target cell sensitization in a standard ⁵¹Cr release assay. Specificity of the effector populations is indicated above each data set.

Figure 4

Testing stop codon readthrough as a means of cryptic epitope expression. (a) Positioning of in-frame stop codons in full-length NP. Either a weak or a strong termination codon (25) was inserted at the BstBI site in NP, between the NP_147–155 and NP_366–374 epitopes. The resulting constructs were recombined into vac. (b) In-frame epitopes encoded after stop codons are not presented. The constructs depicted in a and the indicated control recombinants were tested for target cell sensitization in a standard ⁵¹Cr release assay. Specificity of the effector populations is indicated above each data set.

Figure 4

Testing stop codon readthrough as a means of cryptic epitope expression. (a) Positioning of in-frame stop codons in full-length NP. Either a weak or a strong termination codon (25) was inserted at the BstBI site in NP, between the NP_147–155 and NP_366–374 epitopes. The resulting constructs were recombined into vac. (b) In-frame epitopes encoded after stop codons are not presented. The constructs depicted in a and the indicated control recombinants were tested for target cell sensitization in a standard ⁵¹Cr release assay. Specificity of the effector populations is indicated above each data set.