Inhibition of NF-κB Activation In Vivo Impairs Establishment of Gammaherpesvirus Latency

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Abstract
A critical determinant in chronic gammaherpesvirus infections is the ability of these viruses to establish latency in a lymphocyte reservoir. The nuclear factor (NF)-κB family of transcription factors represent key players in B-cell biology and are targeted by gammaherpesviruses to promote host cell survival, proliferation, and transformation. However, the role of NF-κB signaling in the establishment of latency in vivo has not been addressed. Here we report the generation and in vivo characterization of a recombinant murine gammaherpesvirus 68 (γHV68) that expresses a constitutively active form of the NF-κB inhibitor, IκBαM. Inhibition of NF-κB signaling upon infection with γHV68-IκBαM did not affect lytic replication in cell culture or in the lung following intranasal inoculation. However, there was a substantial decrease in the frequency of latently infected lymphocytes in the lung (90% reduction) and spleens (97% reduction) 16 d post intranasal inoculation. Importantly, the defect in establishment of latency in lung B cells could not be overcome by increasing the dose of virus 100-fold. The observed decrease in establishment of viral latency correlated with a loss of activated, CD69hi B cells in both the lungs and spleen at day 16 postinfection, which was not apparent by 6 wk postinfection. Constitutive expression of Bcl-2 in B cells did not rescue the defect in the establishment of latency observed with γHV68-IκBαM, indicating that NF-κB–mediated functions apart from Bcl-2–mediated B-cell survival are critical for the efficient establishment of gammaherpesvirus latency in vivo. In contrast to the results obtained following intranasal inoculation, infection of mice with γHV68-IκBαM by the intraperitoneal route had only a modest impact on splenic latency, suggesting that route of inoculation may alter requirements for establishment of virus latency in B cells. Finally, analyses of the pathogenesis of γHV68-IκBαM provides evidence that NF-κB signaling plays an important role during multiple stages of γHV68 infection in vivo and, as such, represents a key host regulatory pathway that is likely manipulated by the virus to establish latency in B cells. A central aspect of chronic infection of a host by herpesviruses is the ability of these viruses to establish a quiescent infection (latent infection) in some cell type(s) in which there is only intermittent production of progeny virus (virus reactivation). The establishment of a latent infection in the antibody producing cells of the host immune system (B lymphocytes) is critical for life-long persistence of gammaherpesviruses, as well as the development of virus-associated lymphoproliferative diseases (e.g., B-cell lymphomas). Nuclear factor (NF)-κB transcription factors are a family of cellular proteins that play an important role regulating gene expression in B cells, and it has been shown that gammaherpesviruses have evolved multiple strategies for manipulating NF-κB activity. However, to date there has been no reported examination of the role of NF-κB in the establishment of chronic gammaherpesvirus infection in vivo. Murine gammaherpesvirus 68 (γHV68) infects rodents and shares genetic and biologic properties with the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma–associated herpesvirus. To selectively block the function of NF-κB in infected cells, we engineered a transgenic virus that expresses a repressor of NF-κB activation (IκBαM). Notably, this recombinant virus was defective in the establishment of latency in B cells in the lungs and spleen following intranasal inoculation. We also observed that the decrease in B-cell infection could not be rescued by forced expression of the cellular Bcl-2 protein, which is normally upregulated by NF-κB and serves to protect B cells from some forms of cell death. Thus, we conclude that NF-κB is an important host factor for the successful establishment of a chronic infection by gammaherpesviruses, and likely requires functions of NF-κB apart from its role in B-cell survival.