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Establishment of murine gammaherpesvirus latency in B cells is not a stochastic event.

ABSTRACT: Murid ?-herpesvirus-4 (MuHV-4) promotes polyclonal B cell activation and establishes latency in memory B cells via unclear mechanisms. We aimed at exploring whether B cell receptor specificity plays a role in B cell susceptibility to viral latency and how this is related to B cell activation. We first observed that MuHV-4-specific B cells represent a minority of the latent population, and to better understand the influence of the virus on non-MuHV-4 specific B cells we used the SWHEL mouse model, which produce hen egg lysozyme (HEL)-specific B cells. By tracking HEL+ and HEL- B cells, we showed that in vivo latency was restricted to HEL- B cells while the two populations were equally sensitive to the virus in vitro. Moreover, MuHV-4 induced two waves of B cell activation. While the first wave was characterized by a general B cell activation, as shown by HEL+ and HEL- B cells expansion and upregulation of CD69 expression, the second wave was restricted to the HEL- population, which acquired germinal center (GC) and plasma cell phenotypes. Antigenic stimulation of HEL+ B cells led to the development of HEL+ GC B cells where latent infection remained undetectable, indicating that MuHV-4 does not benefit from acute B cell responses to establish latency in non-virus specific B cells but relies on other mechanisms of the humoral response. These data support a model in which the establishment of latency in B cells by ?-herpesviruses is not stochastic in terms of BCR specificity and is tightly linked to the formation of GCs.

SUBMITTER: Decalf J

PROVIDER: S-EPMC4117635 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Establishment of murine gammaherpesvirus latency in B cells is not a stochastic event.

Decalf Jérémie J Godinho-Silva Cristina C Fontinha Diana D Marques Sofia S Simas J Pedro JP

PLoS pathogens 20140731 7

Murid γ-herpesvirus-4 (MuHV-4) promotes polyclonal B cell activation and establishes latency in memory B cells via unclear mechanisms. We aimed at exploring whether B cell receptor specificity plays a role in B cell susceptibility to viral latency and how this is related to B cell activation. We first observed that MuHV-4-specific B cells represent a minority of the latent population, and to better understand the influence of the virus on non-MuHV-4 specific B cells we used the SWHEL mouse model ...[more]

PMID: 25079788

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Project description: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.

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.

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Establishment of murine gammaherpesvirus latency in B cells is not a stochastic event.

Publications

Establishment of murine gammaherpesvirus latency in B cells is not a stochastic event.

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