IKKα-Mediated Noncanonical NF-κB Signaling Is Required To Support Murine Gammaherpesvirus 68 Latency In Vivo

Noncanonical NF-kappa B signaling is activated in B cells via the tumor necrosis factor (TNF) receptor superfamily members CD40, lymphotoxin beta receptor (LT beta R), and B-cell-activating factor receptor (BAFF-R). The noncanonical 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 noncanonical NF-kappa B signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of I kappa B kinase alpha (IKK alpha), named IKK alpha-SA, with S176A and S180A mutations that prevent phosphorylation by NF-kappa B-inducing kinase (NIK). We controlled for the transgene insertion by introducing two all-frame stop codons into the IKK alpha-SA gene. The IKK alpha-SA mutant but not the IKK alpha-SA.STOP control virus impaired LT beta R-mediated activation of NF-kappa B p52 upon fibroblast infection. IKK alpha-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKK alpha-SA mutant was severely defective in the colonization of the spleen and in the establishment of latency compared to the IKK alpha-SA.STOP control and wild-type (WT) MHV68 at 16 days postinfection (dpi). Reactivation was undetectable in splenocytes infected with the IKK alpha-SA mutant, but reactivation in peritoneal cells was not impacted by IKK alpha-SA. Taken together, the noncanonical NF-kappa B signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68. IMPORTANCE The latency programs of the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block noncanonical NF-kappa B signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKa that is nonresponsive to upstream activation. Latency was reduced in a route-and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the noncanonical NF-kappa B signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.