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Understanding the correlates of immune protection against human immunodeficiency virus and simian immunodeficiency virus (SIV) will require defining the entire cellular immune response against the viruses. Here, we define two novel translation products from the SIV env mRNA that are targeted by the T-cell response in SIV-infected rhesus macaques. The shorter product is a subset of the larger product, which contains both the first exon of the Rev protein and a translated portion of the rev intron. Our data suggest that the translation of viral alternate reading frames may be an important source of T-cell epitopes, including epitopes normally derived from functional proteins.
The pathway from viral infection to the cellular immune response is not well understood. Despite the importance of T-cell responses in control of AIDS virus replication (1, 3, 8, 22), the sources of the peptides recognized by virus-specific T cells are still being discovered. AIDS virus-specific CD8+ T lymphocytes (CD8-TL) recognize complexes of major histocompatibility complex (MHC) class I and virus-derived epitopes presented on the surface of infected cells. These epitopes can be derived from exogenous viral proteins in the infecting virion (19, 20) or from de novo synthesis of viral proteins (9, 21). Additional sources of epitopes are also being explored (4, 6).
CD8-TL can also recognize epitopes derived from translation of viral alternate reading frames (ARFs). Though CD8-TL specific for ARF-derived epitopes have been detected in human immunodeficiency virus (HIV) (2), they remain a largely unexplored source of epitopes that might elicit potent antiviral cellular immune responses. We recently showed that SIVmac239-infected rhesus macaques that spontaneously controlled viral replication, termed elite controllers, made immunodominant CD8-TL responses against an epitope (RHLAFKCLW, or cRW9) derived from an ARF of the env gene (15). This response selected for viral escape in vivo and suppressed viral replication in an in vitro assay. These findings imply that CD8-TL specific for ARF-derived epitopes might be an important component of the total AIDS virus-specific cellular immune response.
Here, we show that the cRW9 epitope is translated as part of two distinct products that differ in size due to start codon usage. The larger and more frequent product contains both the first 23 amino acids of the Rev protein (exon 1) and 50 amino acids translated from the rev intron. The smaller is produced by translation initiation at a start codon within the rev intron and is a subset of the larger product. Finally, we show that these products are degraded after translation from the mature Env-encoding mRNA.
The extent of mRNA splicing is likely a critical determinant of the timing of both viral protein production and epitope recognition. The most rapidly recognized epitopes (excluding those derived from proteins present in the incoming virion) are those derived from proteins encoded by the fully spliced nef and tat (20) and rev (J. B. Sacha, unpublished data) transcripts. In contrast, epitopes derived from translation of partially (or singly) spliced transcripts, such as env, are recognized much later, at 18 h postinfection (19). Consequently, two very different hypotheses emerged as to the origin of cRW9. Since it lies in frame with Rev exon 1 (an early protein), it might be presented early, perhaps within a splice variant form of Rev. However, it also lies in an ARF of Env (a late protein) (Fig. (Fig.1A1A).
We used the kinetic intracellular cytokine staining (KICS) assay that we developed previously (19) to examine the timing of cRW9 recognition. We found that cRW9 was consistently recognized late, 18 h after infection, similarly to an Env epitope, FW9 (Fig. (Fig.1B).1B). Thus, a partially spliced transcript likely encodes cRW9.
To test this hypothesis, we devised a PCR strategy that selectively amplified nonspliced and partially spliced transcripts. We then synthesized cDNA from simian immunodeficiency virus (SIV)-infected cells and used gene-specific PCR, cloning, and sequencing techniques to determine the presence of a specific mRNA that might uniquely encode the product that contains cRW9. As expected, our PCR strategy identified several partially spliced transcripts (data not shown) but all of the transcripts that we found were previously recognized to encode specific proteins (18, 23). However, closer examination of the Env-encoding mRNA, both from our sequences and from those of others (18, 23), revealed that it was a possible source of the cRW9 epitope (Fig. (Fig.2).2). We excluded the first potential start codon downstream of the splice acceptor site (at the junction of the 5′ untranslated region [UTR] and the coding region) as it would encode only a 2-amino-acid product prior to the ribosome encountering the subsequent stop codon. The second start codon is the rev start codon, while the env start codon lies 74 nucleotides downstream. There is also a potential start codon within the rev intron, which lies just 3 nucleotides downstream of the env start and is in frame with both Rev exon 1 and the cRW9 epitope.
Since cRW9 lies in frame with rev exon 1 but is translated from the rev intron (15), we reasoned that cRW9 might be translated from the env mRNA, using either the rev start codon or the start codon in the rev intron, upstream of the cRW9 epitope (Fig. (Fig.2).2). To test these hypotheses, we used site-directed mutagenesis to create mutant versions of SIVmac239 that either knocked out the cryptic start codon (SIV-A6865C) or prevented translation from Rev exon 1 into the intron, by the introduction of a stop codon (SIV-6860insA) or both (SIV-dblmut). We could not knock out the rev start codon directly as this would likely lead to a completely impotent virus.
Next, we synchronously infected MHC class I-matched CD4+ T cells from SIV-naïve macaques with either wild-type SIVmac239 or the mutant viruses. Using the KICS assay, we tested whether these cells could present the cRW9 epitope to a cRW9-specific CD8-TL clone. Surprisingly, we found that both singly mutated viruses were able to present the cRW9 epitope at 18 h postinfection. However, recognition of the SIV-6860insA virus-infected cells was approximately half of that of wild-type-infected cells, while recognition of the SIV-A6865C virus-infected cells was nearly identical to that of wild-type-infected cells. In contrast, recognition was totally ablated when cells were infected with the SIV-dblmut virus (Fig. (Fig.3B).3B). As a positive control, we tested whether cells infected with the various viruses were capable of presenting the Env-FW9 epitope. An Env-FW9-specific T-cell line recognized all cells, indicating that normal viral protein production was not impacted by the introduced mutations (Fig. (Fig.3C).3C). Together, these results imply that the cRW9 epitope is derived from two distinct translational products, Env ARF protein 1 (eARF-P1) and eARF-P2, the more frequent of which (eARF-P1) is produced when the ribosome initiates translation at the Rev start codon on the env mRNA and continues into the rev intron. The smaller product is translated when the ribosome initiates translation at the cryptic AUG codon within the intron (Fig. (Fig.3d3d).
These results strongly suggest that the env mRNA is polycistronic and encodes the Env protein and both eARF-P1 and eARF-P2. To test this hypothesis more directly, we used overlap extension PCR (24) and in vitro transcription to create an env mRNA that contained all of the salient features of the env mRNA produced in infected cells. Specifically, this mRNA contained the same portion of the 5′ UTR present in natural env mRNA, spliced to the coding region in precisely the same way that occurs in the natural env mRNA. Though no particular function has been attributed to this noncoding RNA, it may play an important role in regulation of translation, and so we deemed it necessary to include it in our mRNA.
We next cultured dendritic cells (DCs) from monocytes (7). These cells were derived from SIV-naïve macaques that expressed (or did not express) the following MHC molecules: Mamu-B*17, Mamu-B*08, and Mamu-DPB1*06 (Fig. (Fig.4A).4A). After 5 days in culture, we transfected these cells with the in vitro-transcribed env mRNA using nucleofection. Twelve hours after transfection, we tested whether epitope-specific T-cell lines or clones could recognize the transfected cells. Epitopes derived from all three sources were presented on cells transfected with the env mRNA (Fig. (Fig.4B).4B). A cRW9-specific clone recognized the transfected cells (Fig. (Fig.4B),4B), as did an Env-FW9-specific line (Fig. (Fig.4B)4B) and a Rev-RT11-specific clone (Fig. (Fig.4B),4B), verifying that this single mRNA was translated in both reading frames. As expected, the Rev-RL8 epitope, contained in exon 2, was not recognized (Fig. (Fig.4B).4B). Together, these data verify that the env mRNA was the source of the eARF-P1/P2 products. We also tested whether cells transfected with an mRNA lacking the 5′ UTR were recognized by cRW9- and FW9-specific CD8-TL lines. Recognition of these cells was similar to those of cells transfected with mRNA with the UTR, albeit reduced slightly.
Our data suggest that inefficient ribosomal scanning along the env mRNA might be responsible for translation of eARF-P1/P2. In fact, the rev start codon is a suboptimal Kozak start site (rev start is AUAUGUUAUGA; Kozak consensus is GCC[A/G]CCAUGG [10-12]), which might facilitate ribosomal scanning past the rev start codon to enhance Env translation. These data also provide a scaffold upon which a more-directed search for T-cell epitopes derived from ARFs can be conducted.
Our data do not address the possible functions (or lack thereof) of the eARF-P1/P2 products. They may represent novel viral proteins, or they may be defective ribosomal products without function (25-27). Thus far, attempts to create antibodies against these products have been unsuccessful, likely due to the abundance of hydrophobic amino acids. The SIV-dblmut virus (which knocks out production of these products) replicated as well as did the wild type in vitro (data not shown), indicating that these products are dispensable for in vitro replication. However, it is not known whether this virus can replicate in vivo. Importantly, our data also demonstrate that T-cell epitopes contained within functional proteins can be derived from novel, and possibly aberrant, translation of only a portion of the total protein and from the “wrong” mRNA. Finally, they show that the details of viral gene expression can have unexpected immunological consequences. Understanding these details might enhance our understanding of the total AIDS virus-specific cellular immune response.
We are grateful to S. Lank for sequencing, A. T. Bean for help with cell culture, and E. A. Scheef for continued support. Interleukin 2 used in T-cell culture was kindly provided by the NIH AIDS Reference and Reagent Program (Germantown, MD).
This work was supported by NIH grants R01 AI052056, R01 AI049120, and R24 RR015371 to D.I.W. and grant number P51 RR000167 from the National Center for Research Resources (NCRR), a component of the NIH, to the Wisconsin National Primate Research Center, University of Wisconsin—Madison. This research was conducted in part at a facility constructed with support from Research Facilities Improvement Program grant numbers RR15459 01 and RR020141-01.
This publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.
Published ahead of print on 15 July 2009.