Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection.

The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as "quasi-enveloped" particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2S results from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2S secretion via the secretory pathway. Unlike ORF2C, ORF2S is glycosylated and exists as a dimer. Nonetheless, ORF2S exhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2S does not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.

Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences.

CD8+ cytotoxic T lymphocyte (CTL)-mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

Targeting of the transcription factor STAT4 by antisense phosphorothioate oligonucleotides suppresses collagen-induced arthritis.

The transcription factor STAT4 mediates signals of various proinflammatory cytokines, such as IL-12, IL-15, and IL-23, that initiate and stabilize Th1 cytokine production. Although Th1 cytokine production has been suggested to play a major pathogenic role in rheumatoid arthritis, the role of STAT4 in this disease is poorly understood. In this study, we demonstrate a key functional role of STAT4 in murine collagen-induced arthritis (CIA). In initial studies we found that STAT4 expression is strongly induced in CD4(+) T cells and to a lesser extent in CD11b(+) APCs during CIA. To analyze the role of STAT4 for arthritis manifestation, we next investigated the outcome of interfering with STAT4 gene expression in CIA by using STAT4-deficient mice. Interestingly, STAT4-deficient mice developed significantly less severe arthritis than wild-type control mice and the T cells from such mice produced less IL-6, TNF, and IL-17. In addition, the targeting of STAT4 expression by a specific antisense phosphorothioate oligonucleotide directed at the translation start site suppressed STAT4 levels and signs of CIA even when applied during the onset of disease manifestation. These data suggest a key regulatory role of STAT4 in the pathogenesis and manifestation of murine collagen-induced arthritis. Furthermore, the targeting of STAT4 emerges as a novel approach to therapy for chronic arthritis.

C/EBP alpha and Ets protein family members regulate the human myeloid IgA Fc receptor (Fc alpha R, CD89) promoter.

Fc alpha R (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the Fc alpha R 5'-flanking region from -102 to -64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at -74 and -92, respectively. EMSAs and cotransfection experiments show that C/EBP alpha acts as a major activator of the Fc alpha R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP alpha at -139 and -127. On the other hand, the Fc alpha R Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces Fc alpha R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc alpha R is still incompletely defined, these findings reveal the features controlling the Fc alpha R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc alpha R gene expression associated with its potential roles.

A non-AUG-defined alternative open reading frame of the intestinal carboxyl esterase mRNA generates an epitope recognized by renal cell carcinoma-reactive tumor-infiltrating lymphocytes in situ.

A number of Ags recognized by tumor-reactive T cells have been characterized, including nonmutated gene products and a variety of epitopes shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with an HLA-B7-restricted renal cell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphocytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV complementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (ORF) (a +1 frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence-based peptide binding assay using transfected T2 cells. Constitutive expression of this alternative ORF protein was observed in all transformed HLA-B7+ renal cell lines that were recognized in cytotoxicity assays by the TILs. The intestinal carboxyl esterase gene is transcribed in renal cell carcinoma tumors as well as in normal liver, intestinal, or renal tissues. Mutation of the natural ATG translation initiation site did not alter recognition, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three AUG codons that may be used as alternative translation initiation sites in the +1 ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of the alternative ORF. These results further extend the types of Ags that can be recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion).