Investigation of the infection of endothelial cells with measles viruses and the antiviral mechanism induced by interferon

Das Masernvirus (MV) gehört zu den negativ-strängigen RNA-Viren der Familie der Paramyxoviridae und verursacht beim Menschen akute und subakute Enzephalitiden. Es wurde beschrieben, dass sich MV-RNA in den Endothelzellen von SSPE (subakute sklerosierende Panenzephalitis)-Gehirnen nachweisen lässt (Cosby & Brankin, 1995). In dieser Arbeit konnte ich eine CD46- und CD150-unabhängige Infektion von Endothelzellen durch Wildtyp-MV nachweisen. Ferner wurde beschrieben, dass das Typ II-Interferon (IFN-g) im Serum von Patienten mit akuten Masern und nach einer Masernimpfung erhöht ist (Okada et al., 2001; Ovsyannikova et al., 2003) und dieses Zytokin lässt sich auch in Gehirnläsionen von SSPE-Patienten detektieren (Nagano et al., 1994). Basierend auf diesen Erkenntnissen, konnte ich eine durch das Enzym Indolamin 2,3-Dioxygenase (IDO) vermittelte antivirale Aktivität von IFN-g gegen MV nachweisen. Endothelzellen (EZ) sind bei der akuten Masernerkrankung oder nachfolgenden Komplikationen, die auf einer persistierenden Infektion basieren, wichtige Zielzellen. CD46 und CD150 (signalling lymphocytic activation molecule, SLAM) wurden als zelluläre Rezeptoren für MV beschrieben (Dörig et al., 1993; Naniche et al., 1993; Tatsuo et al., 2000). Es konnte gezeigt werden, dass humane EZ aus dem Gehirn und aus der Nabelschnurvene (HBMECs und HUVECs) zwar CD46, aber auf RNA- und auf Proteinebene kein SLAM exprimieren. Diese Zellen konnten jedoch mit den Wildtyp-MV, die CD46 nicht als Rezeptor benutzen, infiziert werden. Diese Untersuchungen deuten auf die Präsenz eines zusätzlichen Rezeptors für die Aufnahme und Verbreitung von MV in humanen EZ hin. Der antivirale Effekt von Interferonen spielt bei der MV-Vermehrung eine entscheidende Rolle und variiert jedoch in Abhängigkeit von der Wirtszelle (Schnorr et al., 1993). Im Gegensatz zu den attenuierten MV-Impfstämmen können Wildtyp-MV den antiviralen Effekt von Typ I-IFN blockieren, indem sie die Induktion von IFNa/b hemmen und die Sensivität gegenüber dem antiviralen Effekt vermindern. Dabei spielen die V- und C-Proteine des MV eine Rolle (Naniche et al., 2000; Patterson et al., 2000; Shaffer et al., 2003), die mit zellulären STAT-Proteinen und IRF-9 interagieren (Palosaari et al., 2003; Takeuchi et al., 2003; Yokota et al., 2003). In dieser Arbeit konnte gezeigt werden, dass IFN-g die Replikation aller MV-Stämme vorwiegend in Endo- und Epithelzellen hemmen kann und, dass diese durch IFN-g induzierte, antivirale Aktivität mit der Induktion der Indolamin 2,3-Dioxygenase (IDO) korreliert. IDO ist ein Enzym, welches in Anwesenheit von Sauerstoff den Abbau von Tryptophan zu Kynurenin katalysiert (Hirata et al., 1975) und hauptsächlich antiparasitäre, antibakterielle und antivirale (Bodaghi et al., 1999; Adams et al., 2004) Effekte vermittelt. Im Zusammenhang mit Masern wurde beschrieben, dass die Tryptophan Katabolite in SSPE-Patienten erhöht sind (Kurup & Kurup, 2002). Die Daten in dieser Arbeit zeigen, dass die durch IFN-g-induzierte antivirale Aktivität durch Zugabe von L-Tryptophan nahezu aufgehoben werden kann und daher IDO im Zuge der anti-MV Aktivität eine entscheidende Rolle spielt.Measles virus (MV) belongs to the negative-stranded RNA-viruses of the family Paramyxoviridae and causes acute and subacute encephalitis in humans. It has been described that MV-RNA can be detected in endothelial cells of SSPE (subacute sclerosing panencephalitis)-brains (Cosby & Brankin, 1995). In this work I could show a CD46- and CD150-independent endothelial cell infection with wild-type measles viruses. Furthermore it has been described that after acute infections and vaccinations, the type II-interferon (IFN-g) concentrations are increased (Okada et al., 2001; Ovsyannikova et al., 2003) and this cytokine can also be detected in brain lesions of patients suffering from SSPE (Nagano et al., 1994). Based upon this findings I could detect an indoleamine 2,3-dioxygenase (IDO) mediated anti-MV activity of gamma interferon. Endothelial cells (ECs) are important target cells during acute measles and complications following the infection. CD46 and CD150 (signalling lymphocytic activation molecule, SLAM) have been described as cellular receptors for MV (Dörig et al., 1993; Naniche et al., 1993; Tatsuo et al., 2000). It has been shown that human ECs from brain and umbilical vein (HBMECs and HUVECs) were CD46-positive, but did not express SLAM neither at RNA- nor at protein level. However, these cells could be infected with the wild-type MV strains, which do not use CD46 as a receptor. These findings suggest the presence of an additional receptor for MV uptake and spread in human ECs. The antiviral effect of interferons plays an important role for the MV-replication. However, this effect depends from the host cell (Schnorr et al., 1993). Attenuated MV strains are more sensitive to type I-interferons than wild-type strains, because wild-type strains can block the induction of IFN-a/b. The V- and C-proteins of MV play a role in this process (Naniche et al., 2000; Patterson et al., 2000; Shaffer et al., 2003). They interact with STAT proteins and IRF-9 (Palosaari et al., 2003; Takeuchi et al., 2003; Yokota et al., 2003). In this work it could be shown, that IFN-g can inhibit the replication of all MV strains preferably in endo- and epithelial cells. Furthermore it could be demonstrated that the antiviral activity induced by IFN-g correlates with the induction of indoleamine 2,3-dioxygenase (IDO). IDO is an enzyme which in the presence of oxygen catalyses the degradation of tryptophan (Hirata et al., 1975) and is known to mediate antiparasitic as well as antibacterial and antiviral effects (Bodaghi et al., 1999; Adams et al., 2004). In the context with measles it has been described that the tryptophan catabolites were increased in SSPE-patients (Kurup & Kurup, 2002). The data in this work show that the IFN-g-induced antiviral activity can be overcome by the addition of L-tryptophan which indicates a decisive role of IDO in the anti-MV activity

Indoleamine 2,3-Dioxygenase Mediates Cell Type-Specific Anti-Measles Virus Activity of Gamma Interferon

Gamma interferon (IFN-γ) has been shown to be increased in sera from patients with acute measles and after vaccination, to exhibit protective functions in brains of patients with subacute sclerosing panencephalitis, and to mediate a noncytolytic clearance of measles virus (MV) from rodent brains. In order to reveal a possible intracellular antiviral activity in the absence of antigen presentation and cytotoxic T cells, we investigated IFN-γ-induced effects on MV replication in various tissue culture cells. While attenuated MV strains are more sensitive to IFN-α/β than are wild-type strains, IFN-γ inhibits the replication of all MV strains in epithelial, endothelial, and astroglial cells, but not in lymphoid and neuronal cell lines. The antiviral activity induced by IFN-γ correlates with the induction of indoleamine 2,3-dioxygenase (IDO), an enzyme of the tryptophan degradation pathway known to mediate antiviral as well as antibacterial and antiparasitic effects. The IFN-γ-induced antiviral activity can be overcome by the addition of excess amounts of l-tryptophan, which indicates a specific role of IDO in the anti-MV activity. Our data suggest that the IFN-γ-induced enzyme IDO plays an important antiviral role in MV infections of epithelial, endothelial, and astroglial cells

Analysis of cotton rat CD150 expression and tissue distribution.

<p>The presence of cotton rat CD150 was determined on spleen cells and lymph node cells stimulated with Concanavalin A for 24 hours (B and D), and ex vivo leukocytes from spleen (A), thymus (C), Peyer’s patches (F) and lymph node (E) (anti-CD150 black line; grey area isotype control).</p

MV infection of CD150 expressing 293T cells.

<p>(A) HEK 293T and 293T-CrCD150 cells were infected with 50 pfu/well of wild type MV (Bilthoven strain) and plaques were counted 48 hours post-infection. The difference in the number of virus induced plaques was statistically significant between both cell types (p<0.001, ANOVA). (B) After infection of 293T-HuCD150 and 293T-CrCD150 with MV (Bilthoven strain) viral titers were measured at different time intervals. Infection with wild type MV (Bilthoven strain) caused the formation of numerous small viral plaques on 293T-CrCD150 cells (C) and large plaques on 293T-HuCD150 cells (D) (40x magnification).</p

Intranasal Administration of Alpha Interferon Reduces Seasonal Influenza A Virus Morbidity in Ferrets▿

The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, we assessed the protective potential of exogenous IFN-α against seasonal and highly pathogenic influenza viruses in ferrets. Intranasal treatment with IFN-α several hours before infection with the H1N1 influenza A virus strain A/USSR/90/77 reduced viral titers in nasal washes at least 100-fold compared to mock-treated controls. IFN-treated animals developed only mild and transient respiratory symptoms, and the characteristic fever peak seen in mock-treated ferrets 2 days after infection was not observed. Repeated application of IFN-α substantially increased the protective effect of the cytokine treatment. IFN-α did not increase survival after infection with the highly pathogenic H5N1 avian influenza A virus strain A/Vietnam/1203/2004. However, viral titers in nasal washes were significantly reduced at days 1 and 3 postinfection. Our study shows that intranasal application of IFN-α can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. Based on these results, a more intensive evaluation of IFN-α as an emergency drug against pandemic influenza A is warranted

Infectivity of VSV pseudotype viruses on 293T cells expressing cotton rat and human CD150.

<p>(A) HEK 293T cells were stably transfected with plasmids expressing cotton rat (293T-CrCD150) or human CD150 (293T-HuCD150). Expression of CD150 was verified with antibodies against the cotton rat or human CD150 molecule, respectively. (B) 293T, 293T-CrCD150 and 293T-HuCD150 cells were infected with VSVΔG*-Ed-H/F. The infectivity titer was calculated by counting the number of GFP expressing cells. There was a no statistically significant difference in titer between HEK 293T, 293T-CrCD150 and 293T-HuCD150 cells. (C) 293T, 293T-CrCD150 and 293T-HuCD150 cells were infected with VSVΔG*-WTF-H/F. The infectivity titer was calculated by counting the number of GFP expressing cells. There was a significant increase in titer between HEK 293T, 293T-CrCD150 and 293T-HuCD150 cells (p<0.001, ANOVA).</p

Predicted amino acid sequence alignment of cotton rat, human and mouse CD150.

<p>Spaces (indicated by dashes) denote gaps generated during alignment for optimal sequence comparison. Dark shading represents fully conserved residues, while light shading indicates conserved changes. The predicted signal peptide and transmembrane domain of human CD150 are underlined. Potential N-linked glycosylation sites are circled, four cystine residues predicted to form disulfide bonds are denoted by asterisks and three tyrosine-based switch motifs are boxed.</p