Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Proteasome activity and epitope production are altered by IFN-γ treatment, which leads to a gradual replacement of constitutive proteasomes by immunoproteasomes in vitro. However, a quantitative analysis of changes in the steady state subunit composition of proteasomes during an immune response against viruses or bacteria in vivo has not been reported. Here we show that the infection of mice with lymphocytic choriomeningitis virus or Listeria monocytogenes leads to an almost complete replacement of constitutive proteasomes by immunoproteasomes in the liver within 7 days. Proteasome replacements were markedly reduced in IFN-γ−/− mice, but were only slightly affected in IFN-αR−/− and perforin−/− mice. The proteasome regulator PA28α/β was up-regulated, whereas PA28γ was reduced in the liver of lymphocytic choriomeningitis virus-infected mice. Proteasome replacements in the liver strongly altered proteasome activity and were unexpected to this extent, since an in vivo half-life of 12 days had been previously assigned to constitutive proteasomes in the liver. Our results suggest that during the peak phase of viral and bacterial elimination the antiviral cytotoxic T lymphocyte response is directed mainly to immunoproteasome-dependent T cell epitopes, which would be a novel parameter for the design of vaccines.

Myocarditis is a potentially lethal inflammatory heart disease of children and young adults that frequently leads to dilated cardiomyopathy (DCM). Since diagnostic procedures and efficient therapies are lacking, it is important to characterize the critical immune effector pathways underlying the initial cardiac inflammation and the transition from myocarditis to DCM. We describe here a T-cell receptor (TCR) transgenic mouse model with spontaneously developing autoimmune myocarditis that progresses to lethal DCM. Cardiac magnetic resonance imaging revealed early inflammation-associated changes in the ventricle wall including transient thickening of the left ventricle wall. Furthermore, we found that IFN-γ was a major effector cytokine driving the initial inflammatory process and that the cooperation of IFN-γ and IL-17A was essential for the development of the progressive disease. This novel TCR transgenic mouse model permits the identification of the central pathophysiological and immunological processes involved in the transition from autoimmune myocarditis to DCM.

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 M). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor 125 I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 M) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 M) protein degradation was affected, and the cell cycle was arrested in the G 1 /S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response. The human immunodeficiency virus, type I (HIV-I)1 encodes an aspartic endoprotease that is required for cleavage of the viral gag-pol polyprotein. Inhibition of the HIV-I protease leads to the release of noninfectious virus particles and has thus been the aim of drug development in AIDS therapy (1). Several cleavages performed by the HIV-I protease lie between the amino acids phenylalanine (or tyrosine) and proline. Endoproteolytic cleavages N-terminal of proline residues have not been frequently observed in mammalian proteases, which was the premise for the design of transition state mimetics of the PhePro bond as potential inhibitors of the HIV-I protease (2). Interestingly, an exception to this premise arose from the recent analysis of the cleavage specificity of the 20 S proteasome (3) that quite frequently cleaved polypeptide substrates Nterminal of proline residues (4 -8).A number of HIV-I protease inhibitors have been successfully used in clinical therapy of HIV-I infection (9). Patients treated with highly active antiretroviral therapy consisting of HIV-I protease inhibitors and nucleoside analogues showed a marked decrease in viremia with a simultaneous increase in CD4 ϩ helper T cells after the initiation of treatment. Remarkably, even in patients that (because of resistance of the virus) remained viremic upon highly active antiretroviral therapy, the number of CD4 ϩ helper T cells in peripheral blood increased suggesting that there was a direct effect of highly active antiretroviral thera...