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Inhibition of NF-kappaB 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)-kappaB 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-kappaB 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 (gammaHV68) that expresses a constitutively active form of the NF-kappaB inhibitor, IkappaBalphaM. Inhibition of NF-kappaB signaling upon infection with gammaHV68-IkappaBalphaM 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, CD69(hi) 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 gammaHV68-IkappaBalphaM, indicating that NF-kappaB-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 gammaHV68-IkappaBalphaM 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 gammaHV68-IkappaBalphaM provides evidence that NF-kappaB signaling plays an important role during multiple stages of gammaHV68 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.

SUBMITTER: Krug LT

PROVIDER: S-EPMC1781481 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Inhibition of NF-kappaB activation in vivo impairs establishment of gammaherpesvirus latency.

Krug Laurie T LT Moser Janice M JM Dickerson Shelley M SM Speck Samuel H SH

PLoS pathogens 20070101 1

A critical determinant in chronic gammaherpesvirus infections is the ability of these viruses to establish latency in a lymphocyte reservoir. The nuclear factor (NF)-kappaB 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-kappaB signaling in the establishment of latency in vivo has not been addressed. Here we report the generation and in vivo cha ...[more]

PMID: 17257062

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Project description:UnlabelledUracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (ΔUNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68ΔUNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs.ImportanceHerpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the initial site of acute replication was associated with a substantial delay of latency establishment in the spleen. The levels of host UNG were substantially lower in the lung compared to the spleen, suggesting that herpesviruses encode a viral UNG to compensate for reduced host enzyme levels in some cell types and tissues. These data suggest that intervention at the site of initial replicative expansion can delay the establishment of latency, a hallmark of chronic herpesvirus infection.

Project description:BackgroundSilicosis is a complex lung disease for which no successful treatment is available and therefore lung transplantation is a potential alternative. Tumor necrosis factor alpha (TNFalpha) plays a central role in the pathogenesis of silicosis. TNFalpha signaling is mediated by the transcription factor, Nuclear Factor (NF)-kappaB, which regulates genes controlling several physiological processes including the innate immune responses, cell death, and inflammation. Therefore, inhibition of NF-kappaB activation represents a potential therapeutic strategy for silicosis.Methods/findingsIn the present work we evaluated the lung transplant database (May 1986-July 2007) at the University of Pittsburgh to study the efficacy of lung transplantation in patients with silicosis (n = 11). We contrasted the overall survival and rate of graft rejection in these patients to that of patients with idiopathic pulmonary fibrosis (IPF, n = 79) that was selected as a control group because survival benefit of lung transplantation has been identified for these patients. At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells. Patients with silicosis had poor survival (median survival 2.4 yr; confidence interval (CI): 0.16-7.88 yr) compared to IPF patients (5.3 yr; CI: 2.8-15 yr; p = 0.07), and experienced early rejection of their lung grafts (0.9 yr; CI: 0.22-0.9 yr) following lung transplantation (2.4 yr; CI:1.5-3.6 yr; p<0.05). Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition. In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.ConclusionsAlthough limited by its size, our data support that patients with silicosis appear to have poor outcome following lung transplantation. Experimental data indicate that while the systemic inhibition of NF-kappaB protects from silica-induced lung injury, epithelial cell specific NF-kappaB inhibition appears to aggravate the outcome of experimental silicosis.

Project description:Noncanonical NF-κB signaling is activated in B cells via the tumor necrosis factor (TNF) receptor superfamily members CD40, lymphotoxin β receptor (LTβR), 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-κB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IκB kinase α (IKKα), named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NF-κB-inducing kinase (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 LTβR-mediated activation of NF-κB 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 the colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and wild-type (WT) MHV68 at 16 days postinfection (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 noncanonical NF-κB 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-κB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKα 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-κB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.

Project description:UnlabelledGammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.ImportanceGammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make to in vivo virus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAs in vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.

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Inhibition of NF-kappaB activation in vivo impairs establishment of gammaherpesvirus latency.

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Inhibition of NF-kappaB activation in vivo impairs establishment of gammaherpesvirus latency.

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