Satvir S. Tevethia

SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells

MicroRNAs (miRNAs) are small (∼ 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.

NEW SURFACE ANTIGEN IN CELLS TRANSFORMED BY SIMIAN PAPOVAVIRUS SV40.

Summary Hamster cells transformed by papovavirus SV40 in vitro and in vivo possess new surface antigens that can be detected by the indirect immunofluorescent technic. Antibody against the surface antigens of the SV40-transformed cells fail to react with normal cells from various species including the hamster, with the “spontaneously” oncogenic hamster BHK21 cells, or with hamster cells transformed by adenovirus type 12. Inoculation of hamsters with cells not transformed by SV40 did not elicit synthesis of an antibody capable of reacting with the surface antigens of the cells transformed by the papovavirus.

Oncogenicity and cell transformation by papovavirus SV40: the role of the viral genome.

Publisher Summary This chapter evaluates the available knowledge of simian virus 40 (SV40) oncogenesis and assesses the implications for carcinogenesis by DNA-tumor viruses as a whole. It summarizes different tests that can be employed to detect virus-induced changes in SV40-transformed cells. Inoculation of such cells into susceptible hosts usually results in the production of tumors, the ultimate criterion necessary to establish that malignant transformation has indeed occurred. Fusion or cocultivation of the transformed cell with normal susceptible cells may sometimes succeed in the rescue of infectious virus. The optimum conditions required to elicit the production of virus from a majority of the transformed cells have not yet been established. Transformed cells are usually immune to superinfection by the transforming virus. Nucleic acid hybridization experiments indicate that multiple copies of the SV40 genome is present, probably integrated into the host cell chromosome. Virus-specific mRNA is present in the transformed cells. The extent of the transcription of the viral genome seems to vary from one transformed cell line to the next. No relationships have been established between the extent of transcription in a given transformed cell line and the number and/or magnitude of virus-specific changes of that cell line. A series of in vitro tests may detect a variety of surface changes on the transformed cells. These tests include immunofluorescence, agglutination, cytotoxicity, colony inhibition, and mixed hemadsorption.

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.

ABSTRACT The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2b ) mice is directed against three H2-Db -restricted epitopes, I, II/III, and V, and oneH2-Kb -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 β (TCRβ) repertoire of Tag epitope-specific CD8+ cells revealed that multiple TCRβ variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRβ10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8+T-cell responses is established in vivo.

Immune Defects in 28-kDa Proteasome Activator γ-Deficient Mice

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.

Lymphotoxin α−/− Mice Develop Functionally Impaired CD8+ T Cell Responses and Fail to Contain Virus Infection of the Central Nervous System

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8+ T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin α-deficient mice develop CD8+ T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8+ T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTα possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.

In Vivo Ligation of CD40 Enhances Priming Against the Endogenous Tumor Antigen and Promotes CD8+ T Cell Effector Function in SV40 T Antigen Transgenic Mice

The ability to initiate and sustain CD8+ T cell responses to tumors in vivo is hindered by the development of peripheral T cell tolerance against tumor-associated Ags. Approaches that counter the onset of T cell tolerance may preserve a pool of potentially tumor-reactive CD8+ T cells. Administration of agonist Ab to the CD40 molecule, expressed on APCs, can enhance immunization approaches targeting T lymphocytes in an otherwise tolerance-prone environment. In this report, the effects of anti-CD40 administration on priming of naive CD8+ T cells against an endogenous tumor Ag were investigated. Line 501 mice express the SV40 large T Ag oncoprotein as a transgene from the α-amylase promoter, resulting in the development of peripheral CD8+ T cell tolerance to the H-2-Db-restricted immunodominant epitope I of T Ag by 6 mo of age, before the appearance of osteosarcomas. We demonstrate that naive epitope I-specific TCR transgenic (TCR-I) T cells undergo peripheral tolerance following adoptive transfer into 6-mo-old 501 mice. In contrast, administration of agonistic anti-CD40 Ab led to increased expansion of TCR-I T cells in 501 mice, the acquisition of effector function by TCR-I T cells and the establishment of T cell memory. Importantly, this enhanced priming effect of anti-CD40 administration did not require immunization and was effective even if administered after naive TCR-I T cells had encountered the endogenous T Ag. Thus, anti-CD40 administration can block the onset of peripheral tolerance and enhance the recruitment of functionally competent effector T cells toward an endogenous tumor Ag.

Biology of simian virus 40 (SV40) transplantation antigen (TrAg): VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein)

Abstract Simian virus 40 large T antigen (D2 protein), purified to homogeneity from HeLa cells infected with adenovirus 2-SV40 hybrid virus (Ad2+D2), was tested for the presence of SV40 transplantation rejection antigenic sites by the in vivo transplantation rejection assay and by the in vitro lymphocyte-mediated cytotoxicity assay. The results show that the D2 protein immunized BALB/c mice against a challenge of syngeneic SV40 tumor cells. The D2 protein, upon injection into C57B1/6 mice, also induced the generation of thymus-derived cytotoxic lymphocytes which specifically lysed SV40-transformed C57B1/6 mouse embryo fibroblasts. These results suggest that a product(s) coded by the early SV40 genome is present at the surface of SV40-transformed cells and acts as the transplantation rejection antigen.

Biology of simian virus 40 (SV40) transplantation antigen (TrAg): V. In vitro demonstration of SV40 TrAg in SV40 infected nonpermissive mouse cells by the lymphocyte mediated cytotoxicity assay

Abstract The development of SV40-specific transplantation antigen (TrAg) on the surface of nonpermissive mouse cells infected with SV40 was demonstrated using a sensitive in vitro lymphocyte mediated cytotoxicity assay. The in vitro lymphocyte mediated 51 Cr release assay was shown to be specific for the detection of SV40 TrAg. SV40 TrAg was detected 24 hr after infection of mouse cells with SV40 and high levels of TrAg expression persisted for as long as 96 to 120 hr after virus infection. The development of TrAg on the surface of SV40-infected mouse cells correlated with the synthesis of tumor or T antigen in the nucleus of infected cells. The synthesis and the expression of TrAg at the surface of SV40-infected mouse cells may be an important step in the development of immunological resistance of the cell mediated type in an SV40-inoculated host leading to the elimination of stably transformed cells.

Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen

The distribution of antigenic sites recognized by cytotoxic T lymphocytes (CTL) in the amino terminal half of SV40 T antigen was studied using SV40-specific CTL clones. Spleen cells of C57BL/6 (B6) mice immunized with B6/pSV3T3-20GV cells, which synthesize a truncated SV40 T antigen of amino acids 1-368, were restimulated in vitro with B6/pPVU-5-70K cells expressing SV40 T antigen of amino acids 109-708 and then cloned. The recognition sequence for all 10 CTL clones established mapped in the amino terminal half of SV40 T antigen between amino acids 109 and 271. Fine mapping of these 10 CTL clones defined three distinct antigenic sites. These three sites were abolished by the deletion of SV40 T antigen amino acids 193-211, 220-223, and 220-228, respectively. Additional CTL clones were established from spleen cells of B6 mice immunized with B6-K/S11-S24 cells, which synthesize a SV40 T antigen missing amino acids 127-250. None of these CTL clones reacted with B6/pSV3T3-20GV cells. These CTL clones recognized an antigenic site(s) which mapped in the carboxy terminal half of SV40 T antigen. Our results indicate that the antigenic sites in the amino terminal half of SV40 T antigen are tightly clustered between amino acids 193 and 271 and most probably between 193 and 228.