Gamma-herpesvirus latency is preferentially maintained in splenic germinal center and memory B cells

Abstract

The gamma-herpesviruses are oncogenic B cell lymphotrophic viruses that establish life-long latency in the host. Murine gamma-herpesvirus 68 (MHV-68) infection of mice represents a unique system for analyzing gamma-herpesvirus latency in splenic B cells at different stages of infection. After intranasal infection with MHV-68 we analyzed the establishment of latency 14 days after infection, and the maintenance of latency 3 months after infection in different purified subpopulations of B cells in the spleen. The data show that MHV-68 latency is mainly established in germinal center B cells and that long-term latency is preferentially maintained in two different subsets of isotype-switched B cells, germinal center and memory B cells. Cell cycle analysis indicates that MHV-68 is located in both cycling and resting isotype-switched B cells. Analysis of viral gene expression showed that both lytic and latent viral transcripts were differentially expressed in germinal center and memory B cells during long-term latency. Together, these observations suggested that gamma-herpesviruses exploit the B cell life cycle in the spleen.

Figure 1.

Flow cytometry analysis of MHV-68–infected mice spleen B cells 14 d after infection. (A) Germinal center and resting B cells were purified. CD3⁺, CD11b⁺, and CD11c⁺ cells were gated out and resting B cells were then sorted as B220⁺ PNA^low and germinal center B cells as B220⁺ PNA^high. (B) Resting B cells (B220⁺ PNA^low) isolated 14 d after infection were recovered with a mean purity of 98.15% and the contaminating fraction was 1.5% non-B220⁺ cells and 0.03% B220⁺ PNA^high cells. (C) Germinal center B cells (B220⁺ PNA^high) isolated 14 d after infection were recovered with a mean purity of 98.64% and the contaminating fraction was 0.47% non-B220⁺ cells and 0.83% B220⁺ PNA^low cells.

Figure 2.

MHV-68 establishes latency in the spleen mainly in germinal center B cells. Spleen B cells 14 d after infection were sorted as (A) germinal center B cells and (B) resting B cells. The purified cells were analyzed by two different methods to determine the frequency of cells carrying MHV-68. The frequency of genome-positive cells was determined by LDA-PCR assay (▪). The frequency of infectious virus was determined by infectious virus-LDA (•). The percentages of CPE or PCR reactions that scored positive as a function of the number of cells analyzed are shown. Data represent the average of three to four experiments and the error bars represent SD.

Figure 3.

Flow cytometry analysis of MHV-68–infected spleen cells 3 mo after infection. (A) Spleen B cells were purified as isotype-switched cells (IgA⁺, IgG₁ ⁺, IgG_2ab ⁺, IgG₃ ⁺) and naive cells (IgM/D⁺). (B) Naive B cells (IgM/D⁺) were obtained with a mean purity of 99% and the contaminating fraction was 0.8% non-B cells and 0.02% isotype-switched B cells. (C) Isotype-switched B cells (IgA⁺, IgG₁ ⁺, IgG_2ab ⁺, IgG₃ ⁺) were further separated into memory and germinal center subpopulations on the basis of CD38 expression. (D) Germinal center B cells (isotype-switched CD38^low) were recovered with a mean purity of 96% and the contaminating fraction was 2.23% nonisotype-switched cells and 1.9% memory B cells. (E) Memory B cells (isotype-switched CD38^high) were recovered with a mean purity of 95% and the contaminating fraction was 2.23% nonisotype-switched cells and 2.29% germinal center B cells. (F) PNA expression on germinal center (isotype-switched CD38^low, gray curve) and memory (isotype-switched CD38^high, empty curve) B cells 3 mo after infection. (G) Fas expression on germinal center (isotype-switched CD38^low, gray curve) and memory (isotype-switched CD38^high, empty curve) B cells 3 mo after infection.

Figure 4.

MHV-68 maintains latency in every subset of splenic B cells. Spleen B cells 3 mo after infection were sorted as (A) total B cells, (B) naive B cells, (C) memory B cells, and (D) germinal center B cells. The purified cells were analyzed by two different methods to determine the frequency of cells carrying MHV-68. The frequency of genome-positive cells was determined by LDA-PCR assay (▪). The frequency of infectious virus was determined by infectious virus-LDA (•). The percentages of CPE or PCR reactions that scored positive as a function of the number of cells analyzed are shown. Data represent the average of three to four experiments and the error bars represent SD.

Figure 5.

Detection of MHV-68–infected cells in purified G₀+G₁ and S+G₂/M subpopulations of isotype-switched spleen B cells 3 mo after infection. (A) FACS^® analysis of the DNA content of isotype-switched B cells gated as CD19⁺ IgA⁺ IgG₁ ⁺ IgG_2ab ⁺ IgG₃ ⁺ IgD/M⁻ before the fractionation into G₀+G₁ (94.9% of the population) and S+G₂/M (5.3% of the population). The percentages are the average of two independent experiments. (B) The percentage of isotype-switched cells into G₀+G₁ and S+G₂/M harboring viral genome was assessed by LDA-PCR assay. The analysis shown is representative of two independent experiments, each one pooling seven spleens 3 mo after MHV-68 infection.

Figure 6.

(A) Detection of MHV-68 gene expression in the tumor B cell line S11. Total RNA from S11 cells was subjected to RT-PCR as described in Materials and Methods. (B) Detection of MHV-68 gene expression in purified memory and germinal center B cells. Spleen cells were sorted and subjected to RT-PCR and agarose gel electrophoresis as described in Materials and Methods. Primers specific for MHV-68 ORFs M1, M2, M3, M9, M11, 72, 73, 74, K3, and 50 and for mouse β actin were used as indicated. Control reactions lacking RT are also shown.