Non-canonical NF-kappaB signaling is activated in B cells via TNF receptor superfamily members CD40, Lymphotoxin beta-R, and BAFF-R. The non-canonical pathway is required at multiple stages of B-cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt non-canonical NF-kappaB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IKKalpha, named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NIK. We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTbetaR-mediated activation of NF-kappaB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and WT MHV68 at 16 dpi. Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the non-canonical NF-kappaB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68.
Interplay of Murine Gammaherpesvirus 68 with NF-kappaB Signaling of the Host
