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

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.

Inhibition of NF-κB Signaling Reduces Virus Load and Gammaherpesvirus-Induced Pulmonary Fibrosis

“…23,31 Mice were inoculated intranasally as we have described before with 1 ϫ 10 5 plaque forming units (pfu) of wild-type MHV68, recombinant MHV68-IB␣M (␣M), or a marker rescue of recombinant MHV68-IB␣M (IB␣M-MR) mixed in Dulbecco modified Eagle medium (DMEM)-serum free in a total volume of 20 to 40 l. 17 Supernatant of homogenated uninfected NIH3T12 cells was used to inoculate mock treated mice or naïve mice were used as controls. At the indicated times post infection, mice were sacrificed and bronchoalveolar lavage (BAL) was performed through a tracheal canula by twice instilling then withdrawing 0.6 ml of serum free complete medium (Cellgro, Herndon, VA).…”

“…The NF-B signaling pathway is activated during the productive infection of fibroblasts and latent infection of B cells with MHV68, 23,31 but the activation profile in lung epithelial cells in culture or lung tissue in vivo has not been investigated. To examine the activation profile on de novo infection of murine airway epithelial cells, nuclear extracts were prepared from LA-4 cells infected at a multiplicity of infection (MOI) of 5 pfu per cell and analyzed for binding to an oligonucleotide containing a NF-B binding sites by EMSA.…”

“…26,28,29 We previously generated a recombinant MHV68 that expresses a mutant form of IB␣ (MHV68-IB␣M) that functions as a dominant inhibitor of NF-B signaling. 23 In vivo infection of C57BL/6 mice with this recombinant MHV68 demonstrated that inhibition of NF-B in the host cell during virus infection does not impact virus replication. However, interference with NF-B signaling in infected cells results in deficient establishment of latency.…”

“…Several studies indicate that the gammaherpesvirus life cycle can be regulated through the cellular nuclear factor (NF)-B signaling pathway. 23,24 Expression of the NF-B subunit p65 inhibits lytic replication of MHV68, suggesting that high levels of NF-B promote the establishment of latency. 24 In a resting nonactivated state NF-B dimers are sequestered in the cytoplasm as a result of their association with inhibitory proteins including IB␣.…”

“…However, interference with NF-B signaling in infected cells results in deficient establishment of latency. 23 To determine the role of NF-B signaling in virus latency and virus-induced profibrotic response, we infected IFN-␥R Ϫ/Ϫ mice with MHV68-IB␣M. We found that expression of the super repressor of NF-B in infected cells impaired latency and persistence in the lung and protected mice from fibrosis as evidenced by low collagen deposition and decreased expression of smooth muscle specific-␣ actin (␣SMA) and PAI-1 and the chemokines MCP-1, CXCL12, as well as the profibrotic factor TGF-␤.…”