Pathogen-associated molecular patterns decisively influence antiviral immune responses, whereas the contribution of endogenous signals of tissue damage, also known as damage-associated molecular patterns or alarmins, remains ill defined. We show that interleukin-33 (IL-33), an alarmin released from necrotic cells, is necessary for potent CD8(+) T cell (CTL) responses to replicating, prototypic RNA and DNA viruses in mice. IL-33 signaled through its receptor on activated CTLs, enhanced clonal expansion in a CTL-intrinsic fashion, determined plurifunctional effector cell differentiation, and was necessary for virus control. Moreover, recombinant IL-33 augmented vaccine-induced CTL responses. Radio-resistant cells of the splenic T cell zone produced IL-33, and efficient CTL responses required IL-33 from radio-resistant cells but not from hematopoietic cells. Thus, alarmin release by radio-resistant cells orchestrates protective antiviral CTL responses.

The IL-1 receptor-related protein T1 is expressed on the surface of Th2, but not Th1 cells. Studies with anti-T1 monoclonal antibodies have suggested that T1 is critical for development of normal Th2-type responses. To elucidate the role of T1 in vivo, we generated T1deficient mice and a T1-transgenic strain which secretes soluble T1-Fc fusion protein into the serum. These were analyzed for the Th2 immune response induced by infection with the parasitic nematode Nippostrongylus brasiliensis. Although Th2 cytokine production by lymph node cells was similar in all groups of N. brasiliensis-infected mice, a decrease in IL-5 production by lung lymphocytes was detected in both T1-deficient and T1-Fc-transgenic mice compared to control littermates. This difference in IL-5 production did not influence blood eosinophilia, but recruitment of eosinophils into lung tissue, especially in T1-Fctransgenic mice was slightly decreased. However, induction of all other immune parameters was normal and both T1-deficient and T1-Fc-transgenic mice were able to clear the parasite infection within 12 days with kinetics similar to those in control mice. Therefore, in contrast to previous suggestions, we conclude that the T1 protein is not obligatory for normal development of Th2 immune responses.

Over the past decade, adeno-associated (AAV) virus has emerged as an important vector for gene therapy. As a result, understanding its basic biology, including intracellular trafficking, has become increasingly important. Here, we describe the effect of inhibiting dynein function or altering the state of microtubule polymerization on rAAV2 transduction. Overexpression of dynamitin, resulting in a functional inhibition of the minus-end-directed microtubule motor protein dynein, did not inhibit transduction. Equally, treatment of cells with nocodazole, or concentrations of vinblastine that result in the disruption of microtubules, had no significant effect on transduction. In contrast, high concentrations of Taxol and vinblastine, resulting in microtubule stabilization and the formation of tubulin paracrystals respectively, reduced rAAV2 transduction in a vector-dose-dependent manner. These results demonstrate that AAV2 can infect HeLa cells independently of dynein function or an intact microtubule network.

Exposure of Semliki Forest virus 1 to mildly acidic conditions results in conformational changes of the viral spike proteins, which in turn leads to a pore formation across its membrane. The ability to form a pore has been ascribed to the ectodomain of the Semliki Forest virus (SFV) E1 spike protein. To elucidate whether the E1 protein per se is sufficient for low pH-dependent pore formation, we expressed E1 in Escherichia coli in an inducible manner using the pET11c expression system. The data obtained clearly showed that the E1 protein was expressed in the bacterial cell membrane and that exposure of E. coli expressing the SFV E1 protein to low pH (<6.2) resulted in a permeability change of the membrane. Thus, we conclude that the E1 protein of SFV per se is sufficient to promote pore formation under mildly acidic conditions.The entry of a virus into a host cell is an essential step in the chain of events leading to infection. A multitude of viruses use the endocytotic pathway to access host cells. As a model for the entry of enveloped animal viruses into cells, the ␣ virus Semliki Forest virus has been extensively studied (1). Once attached, the virus is internalized via coated vesicles and transferred to the endosome. Due to the acidic conditions within this organelle, the lipid envelope of SFV fuses with the endosomal membrane of the target cell (2). This low pH-induced fusion is mediated by the so called virus spikes (3-5). Each spike is a heterotrimer, being composed of the type I integral membrane glycoproteins E1 (50.786 kDa) and E2 (51.855 kDa), plus the peripheral glycoprotein E3 (11.369 kDa), which is associated with E2 (6). Several functions have been ascribed to the spike proteins; e.g. the E2 and E3 precursor protein p62 forms a heterodimer with E1 in the endoplasmic reticulum and is responsible for the transport of the complex to the plasma membrane (6). The E1 protein is involved in the acid-induced fusion of the viral and endosomal membranes (7-9).Under mildly acidic conditions (pH 5.8) the spike proteins undergo an irreversible conformational change that results in the dissociation of the E1/E2/E3 complex, the formation of an E1 homotrimer and the exposure of a fusion peptide on the E1 protein (7). This conformational change also leads to the formation of a pore, which causes an alteration in the permeability of the virion membrane or of a cell membrane expressing the spike proteins (10 -14). It has been suggested that this acid-induced pore formation plays a crucial role in the penetration and uncoating process of SFV (15).Several experiments have shown that pore formation is dependent on the ectodomain of the E1 spike protein (14,16,17).It has been speculated that the E1 protein per se would be sufficient for triggering acid-induced pore formation. So far, all attempts to express isolated E1 protein on the surface of eukaryotic cells have failed. The E1 protein was synthesized but retained in the endoplasmic reticulum, since efficient transport of the glycoproteins to the plasma membrane req...

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