MicroRNAs (miRNAs) are small (approximately 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein-Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2 b ) mice is directed against three H2-D b -restricted epitopes, I, II/III, and V, and one H2-K b -restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8 ؉ T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8 ؉ T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8 ؉ T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8 ؉ T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8 ؉ T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor ␤ (TCR␤) repertoire of Tag epitope-specific CD8 ؉ cells revealed that multiple TCR␤ variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCR␤10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8 ؉ T-cell responses is established in vivo.Immunity to the large tumor antigen (Tag) of simian virus 40 (SV40) in C57BL/6 mice is characterized by the development of CD8 (23,34,50,52). An immunological hierarchy has been demonstrated among these four epitopes within Tag. Immunization of C57BL/6 mice with SV40, SV40 Tagtransformed cells, or a recombinant vaccinia virus (rVV) which encodes the full-length Tag leads to the induction of cytotoxic T lymphocytes (CTL) specific for epitopes I, II/III, and IV (26, 41, 51). Frequency estimates from limiting-dilution analysis of splenic lymphocytes obtained 9 days after immunization with SV40 Tag-transformed cells revealed that epitope IV-specific CTL represent 1 in 14,000 splenocytes while epitope I and II/III-specific CTL were less abundant (1 in 67,000) and epitope V-specific CTL were undetectable (41).Although epitope V-specific CTL are not detected following immunization with full-length SV40 Tag, immunization with syngeneic cells carrying inactivating mutations or deletions in Tag epitopes I, II/III, and IV leads to the induction of epitope V-specific CTL (41, 50). Accordingly, epitope V has been characterized as immunorecessive. Additional strategies which enhance the immunogenicity of epitope V include immunization with rVVs which express epitope V as a minigene linked to a secretory signal sequence (ES) or in which the epitope V sequence is inserted into a nonimmunogenic murine self protein, dihydrofolate reductase (26). Precise mechanisms which control the immun...

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-γ-inducible PA28α/β complex in Ag processing. Although the noninducible and predominantly nuclear PA28γ complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28γ-deficient mice and investigated their immune function. PA28γ−/− mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28γ−/− mice, like PA28α−/−/β−/− mice, are deficient in the processing of only specific Ags.