Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression

Das murine Gammaherpesvirus 68 als <em>in vivo</em> Modell zur Evaluierung von Antigen-spezifischen Vakzinen.

Die Entwicklung wirksamer Impfstoffe gegen chronische Infektionskrankheiten wie AIDS oder Hepatitis C wird durch das Fehlen geeigneter Kleintier-Modelle stark beeintr&auml;chtigt. In dieser Arbeit konnte gezeigt werden, dass rekombinantes murines Gammaherpesvirus 68 (MHV-68) als Testsystem geeignet ist, die Potenz Antigen-spezifischer Testvakzine im Kontext einer nat&uuml;rlichen chronischen Infektion zu evaluieren. Nach der Etablierung des Systems mit dem Modell-Antigen Ovalbumin wurden als Antigene einer wichtigen chronischen Virusinfektion des Menschen die Proteine NS3 und CORE des Hepatitis C Virus gew&auml;hlt. Die BAC-Technologie erlaubte die einfache und schnelle Herstellung der rekombinanten MHV-68-OVA, MHV-68-NS3 und MHV-68-CORE. Vorstudien zur Charakterisierung der rekombinanten MHV-68 zeigten, dass die Insertion der Gene eine effiziente Expression der Proteine in Zellkultur erm&ouml;glichte und die Replikationsf&auml;higkeit der Viren in vitro nicht eingeschr&auml;nkt war. Die in vivo Charakterisierung der rekombinanten MHV-68 verdeutlichte, dass zwar die lytischen Titer vergleichbar zum Wildtyp, die latenten Parameter Splenomegalie und Zahl latent infizierter Milzzellen, abh&auml;ngig vom inserierten Antigen, jedoch ver&auml;ndert waren. Am Beispiel von MHV-68-OVA gelang es erstmals, eine Belastungsinfektion im Kontext einer Immunisierung zu etablieren und einen Vakzinierungseffekt durch Messung MHV-68-spezifischer Parameter zu untersuchen. Nach Ovalbumin-spezifischer Immunisierung mit MVA-OVA konnten sowohl die lytische als auch die latente Infektion mit MHV-68-OVA signifikant beeinflusst werden. Genauso effizient wurden die Parameter einer Belastungsinfektion mit MHV-68-NS3 nach Vakzinierung mit MVA-NS3 oder prime-boost Immunisierung mit Ad-NS3/MVA-NS3 verringert. Intrazellul&auml;re Zytokinf&auml;rbungen, Tetramerf&auml;rbungen und 51Cr-Freisetzungs-Assays zeigten, dass CD8+, aktivierte, IFN&gamma;-produzierende T-Lymphozyten an der Antigen-spezifischen Immunantwort und der Reduktion spezifischer Parameter der MHV-68 Infektion beteiligt waren. Gleichzeitig verdeutlichten diese Methoden, dass MHV-68 zudem als Stimulator f&uuml;r die effiziente Expansion Antigen-spezifischer T-Zellen geeignet ist. Die verwendeten Antigene konnten ihre Eigenschaften hinsichtlich ihrer Immunogenit&auml;t auch nach Insertion in MHV-68 beibehalten. Die Ergebnisse der vorliegenden Arbeit zeigen daher die Eignung von MHV-68 als Modell zur Impfstoffevaluierung. Der M&ouml;glichkeit, diverse Vakzine mit verschiedenen Impfstrategien, Applikationsformen und Adjuvantien zu verwenden, sind dabei keine Grenzen gesetzt. Im Vergleich zu bisherigen Mausmodellen stellt MHV-68 eine attraktive Option dar, um Impfstoffe gegen verschiedenste Antigene im Zusammenhang einer akuten sowie chronischen Infektion zu evaluieren

In vivo attenuation of recombinant murine gammaherpesvirus 68 (MHV-68) is due to the expression and immunogenicity but not to the insertion of foreign sequences.

Recombinant herpesviruses are increasingly utilized to study herpesvirus biology. For recombinant viruses carrying insertions of foreign sequences, attenuated phenotypes in vivo have been frequently observed. In most cases, the underlying mechanisms were not clear or have not been investigated. In this study, we used a recombinant murine gammaherpesvirus 68 (MHV-68), carrying a cassette for the expression of the non-structural protein NS3 of Hepatitis C virus (MHV-68-NS3), to systematically address the question whether the insertion of a defined foreign sequence (NS3) interferes with the biological properties of the recombinant virus in vivo, and to analyze the underlying mechanism. We show that while MHV-68-NS3 is attenuated in vivo, recombinant MHV-68 carrying identical genomic inserts but unable to express the NS3 protein, are not attenuated. Moreover, we provide evidence that the attenuated phenotype of MHV-68-NS3 is caused by the immune response. Our findings are important for the in vivo use of recombinant MHV-68 carrying insertions of marker genes, reporter genes or genes of model antigens. They are also relevant for the potential application of MHV-68 as gene delivery vector

Recombinant murine gammaherpesvirus 68 (MHV-68) as challenge virus to test efficacy of vaccination against chronic virus infections in the mouse model.

Efficient vaccines against AIDS, Hepatitis C and other persistent virus infections are urgently needed. Vaccine development has been especially hampered by the lack of suitable small animal models to reliably test the protective capacity of candidate vaccines against such chronic viral infections. A natural mouse pathogen such as MHV-68 that persists lifelong after infection, appears to be a particularly promising candidate for a more relevant model system. Here, we investigated infections with recombinant MHV-68 as novel mouse challenge model to test the efficacy of heterologous vaccines based on recombinant modified vaccinia virus Ankara (MVA). To apply ovalbumin (OVA) as a model antigen, we constructed the recombinant virus MHV-68-OVA by BAC technology and characterized genetic stability and replicative capacity of the virus in vitro and in vivo. We demonstrated the ability of MHV-68-OVA to produce ovalbumin upon tissue culture infection. Moreover, the use of MHV-68-OVA-infected target cells allowed for efficient ex vivo amplification of OVA-specific, MHC class I-restricted CD8 T cells derived from MVA-OVA-vaccinated C57BL/6 mice. Finally, we immunized C57BL/6 mice with MVA-OVA and challenged the animals with MHV-68-OVA testing different time points and routes of infection. Vaccinated mice were infected with MHV-68-OVA but showed reduced viral loads in the acute and latent phase of challenge infection. These data strongly suggest the usefulness of the MHV-68 challenge model for further evaluation of recombinant vaccines against persisting virus infections

A gammaherpesviral internal repeat contributes to latency amplification.

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression

The 37-kDa/67-kDa laminin receptor acts as a receptor for infectious prions and is inhibited by polysulfated glycanes.

BACKGROUND: Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS: For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS: The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS: We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells