We report that various nontraditional viral epitopes are presented by MHC-I molecules in HIV-1–infected cells. We first identified in the HIV-1 genome 3 ARFs potentially encoding polypeptides from 34 to 52 amino acids in length (which we named Gag-ARFP, Pol-ARFP, and Env-ARFP). Various transcriptional and translational mechanisms, such as ribosomal frameshifting or internal AUG translation initiation, may account for the biosynthesis of these nontraditional polypeptides (for review see reference 25
). For example, Gag-ARFP could be produced through a ribosomal frameshifting from the gag
sequence. Although this remains to be formally proven, we noticed the presence of a conserved slippery site (UUUAAAU) upstream of the Gag-ARFP start codon that may promote the frameshifting (39
). Of note, we were unable to detect the presence of Gag-ARFP in HIV-infected cells (unpublished data). This could be due to the poor sensitivity of the anti–Gag-ARFP polyclonal antibodies that we have generated (unpublished data). It is also conceivable that Gag-ARFP, as well as other ARFPs, are short-lived and/or expressed at low levels. These polypeptides could be related to defective ribosomal products that are prematurely terminated or misfolded polypeptides rapidly degraded by the proteasome (40
). Abrogating Gag-ARFP expression by inserting a premature stop codon within the ARF did not affect viral growth, indicating that this small protein is not essential for the viral life cycle. Determining how Gag-ARFP, Pol-ARFP, or Env-ARFP are produced and understanding their biological function will require further investigations.
Using an algorithm, we defined six ARFP-derived epitopes putatively presented by the HLA-B7 molecule. Several lines of evidence indicate that these unconventional epitopes are of immunologic relevance. First, the six peptides that we selected are immunogenic in HLA-B7 mice. The repertoires of epitopes recognized by HLA-B7mα3
mouse and by human T cells are comparable in their breadth (26
); thus, this murine model offers a rapid and convenient system to assess immunogenicity of various antigens, including peptides. Second and most importantly, the six epitopes are generated and recognized in HIV-1–infected patients. This was demonstrated by ex vivo IFN-γ ELISPOT and intracellular labeling of patient PBMCs, as well as by a 51
Cr release test performed after a short period of PBMC stimulation, which confirmed the killing activity of peptide-stimulated cells. With each of the six ARFP-derived peptides, a significant response was observed in ~50% of the HLA-B7+
HIV-infected patients analyzed. The breadth of the response varied between each individual, ranging from 1/6 to 6/6 peptides recognized. Third, we show that the Gag-ARFP–derived epitope Q9VF is generated in vitro in HIV-infected cells because infected cells were recognized by Q9VF-specific CTLs. Moreover, the Gag-ARFP negative mutant HIV strain carrying a premature stop codon was no longer able to activate these CTLs, indicating that Q9VF is generated from the expected ARF. It will be worth determining whether the other ARFP-derived epitopes are similarly presented by HIV-infected cells in culture. Altogether, these results indicate that various nontraditional epitopes are presented by MHC-I molecules in HIV-1–infected cells, leading to the activation of specific CTLs.
A comprehensive analysis of naturally occurring viral sequences in the Los Alamos database revealed various patterns of intraclade and interclade conservation of the six selected peptides. Some peptides are highly conserved (64 and 80% intraclade B conservation for Env-ARFP–derived M9PT and G10QT epitopes, respectively). Others are less frequently encountered, such as Pol-ARFP–derived A9RL and S10PV epitopes (24 and 29% intraclade B conservation, respectively). The Gag-ARFP–derived Q9VF peptide is infrequent among viral isolates, but its cross-reactive variant Q9VF/5N is found in up to 68% of clade B viruses. We have also performed a preliminary analysis of the provirus sequences encountered in three of the HLA-B7+ patients studied here. In agreement with the analysis of the Los Alamos database, we observed in these patients a high variability of Gag-ARFP sequences, which encoded mostly Q9VF/5N, and more rarely Q9VF or other variant epitopes (unpublished data). Altogether, these results indicate that ARFPs may generate either conserved or variable epitopes, similar to primary ORF-encoded epitopes. It is important to note that the observed conservation of a given ARF sequence will depend not only on immunological and virological selection pressures exerted on its ARFP, but also on the protein encoded by the corresponding primary ORF.
We have limited our analysis to epitopes presented by HLA-B7 haplotypes and encoded by sequences beginning with AUG and preceded by a consensus Kozak sequence. One can expect that other nontraditional epitopes may be presented by various HLA molecules. Different cryptic translation mechanisms, such as non-AUG translation initiation (42
), have been reported in other systems and may be operative in HIV-1–infected cells as well (for review see reference 43
). Therefore, the repertoire of viral epitopes is certainly much broader than previously thought (44
). Although these nontypical epitopes may be infrequent, we provide evidence here that they are generated during natural HIV-1 infection. This underappreciated source of epitopes should be studied more extensively (e.g., by longitudinally monitoring CTL responses during the course of HIV infection. Nontypical epitopes should be taken into account when designing candidate vaccine formulations.