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. 2005 Mar;79(5):2891-9.
doi: 10.1128/JVI.79.5.2891-2899.2005.

Establishment and maintenance of long-term murine gammaherpesvirus 68 latency in B cells in the absence of CD40

David O Willer  1 , Samuel H Speck
Affiliations

Establishment and maintenance of long-term murine gammaherpesvirus 68 latency in B cells in the absence of CD40

David O Willer et al. J Virol. 2005 Mar.

Abstract

Murine gammaherpesvirus 68 (gammaHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of gammaHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, gammaHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in gammaHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40(-/-)) mice. Here we report that, consistent with previous observations, gammaHV68 efficiently established a latent infection in B cells of CD40(-/-) mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40(-/-) mice 42 days postinfection was observed. In addition, in contrast to gammaHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of gammaHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that gammaHV68 may either directly infect memory B cells present in CD40(-/-) mice or be capable of driving differentiation of naive CD40(-/-) B cells. A possible explanation for the apparent discrepancy between the failure of gammaHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of gammaHV68 B-cell latency in CD40(-/-) mice came from examining virus replication in the lungs of infected CD40(-/-) mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40(-/-) mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.

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Figures

FIG. 1.
FIG. 1.
Acute-phase γHV68 titers are unaffected by the host mouse strain. C57BL/6J and CD40−/− mice were inoculated with 1,000 PFU of wild-type γHV68, and their lungs were harvested at 4 and 9 dpi. The data shown were compiled from single experiments with 9 or 10 individual mice. Virus titers from lung homogenates were determined by plaque assay on NIH 3T12 monolayers as described in Materials and Methods. Each point represents the virus titer from an individual mouse. The solid line represents the mean virus titer for each group of mice, and the dashed line represents the limit of detection of this assay (50 PFU).
FIG. 2.
FIG. 2.
γHV68 reactivation from latency in CD40−/− mice. Unsorted splenocytes or purified splenic cell populations were obtained from γHV68-infected CD40−/− mice at the times indicated and subjected to analysis for reactivation upon explant culture. Serial dilutions of intact (live) cells were plated onto MEF indicator monolayers in parallel with samples that had been mechanically disrupted. The level of reactivation that could be attributed to preformed infectious virus was either undetectable or very low (data not shown). Best-fit curves were derived from nonlinear regression analysis, and each symbol represents the mean percentage of wells positive for a virus-induced CPE ± the standard error of the mean. The dashed line represents 63.2%, from which the frequency of genome-positive cells or the frequency of cell reactivation of virus was calculated with a Poisson distribution. The data shown represent at least two independent experiments with pooled cells from 10 to 15 mice per experimental group.
FIG. 3.
FIG. 3.
γHV68 splenic latency in CD40−/− mice. Unsorted and FACS-sorted splenocytes were obtained from CD40−/− mice at 16, 42, and 84 dpi. The frequency of viral genome-positive cells within these cell populations was determined by a limiting-dilution PCR assay as described in Materials and Methods. The data shown represent at least two independent experiments with pooled cells from 10 to 15 mice per experimental group. Curve fit lines were derived from nonlinear regression analysis, and symbols represent mean percentages of wells positive for viral DNA. The error bars represent the standard error of the mean. The dotted line represents 63.2%, from which the frequency of viral genome-positive cells was calculated with a Poisson distribution.
FIG. 4.
FIG. 4.
FACS analysis of splenocyte populations in CD40−/− mice. Splenocytes collected from CD40−/− mice were isolated and prepared as described in Materials and Methods. Purification of B-cell and non-B-cell fractions was based on surface expression of the pan-B-cell marker CD19 (data not shown). Purified B cells (CD19+) were further fractionated into naive (IgD+) and IgD− B cells with a fluorescein isothiocyanate-conjugated antibody to IgD. Representative FACS plots from one replicate experiment are shown. (A) Presorting analysis of total splenocytes stained with antibodies directed against CD19 and IgD. (B) Postsorting FACS analysis indicating the purity of sorted lymphocyte subsets. Mean postsorting purities were as follows: CD19+, 97.4% ± 2.0%; CD19−, 99.4% ± 0.9%; CD19+ IgD+, 95.0% ± 5.2%; CD19+ IgD−, 94.8% ± 0.6.%. The contaminating fractions for B-cell and non-B-cell fractions were 2.5 and 0.6%, respectively. Contamination within the naive B-cell fraction was 0.45% IgD− B cells, and contamination within the IgD− B-cell population with naive B cells was 2.5%.
FIG. 5.
FIG. 5.
Persistent γHV68 viral replication in the lungs of CD40−/− mice. Lung homogenates from γHV68-infected CD40−/− and C57BL/6J mice were generated at 42 and 84 dpi as described in Materials and Methods. Serial dilutions of lung homogenates were plated onto MEF indicator monolayers and scored microscopically for the presence of a CPE 10 to 12 days postplating. Twenty-four replicates were plated per dilution. The data represent two replicate experiments with pooled lungs from five mice per experiment.
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