A gammaherpesvirus 68 gene 50 null mutant establishes long-term latency in the lung but fails to vaccinate against a wild-type virus challenge

Abstract

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (gammaHV68). Introduction of a translation stop codon in gammaHV68 gene 50 (gene 50.stop gammaHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop gammaHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop gammaHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type gammaHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

FIG. 1.

Analysis of γHV68 latency following infection with G50.stop. (A to C) Lungs from C57BL/6J mice infected i.n. with either γHV68-CreGFP (WT) or G50.stop (50KO) at day 16 (A), day 42 (B), and 4 months (C) postinfection were harvested, digested with collagenase (type IV; Worthington Biochemical Corporation, Lakewood, NJ), and sorted by magnetic bead cell sorting (MACS; Miltenyi Biotec, Auburn, CA) to enrich for B cells. Bulk spleen cells were also harvested at the same time points. Cells were analyzed by limiting dilution viral genome PCR as previously described (25). Serial dilutions of cells were plated into a background of 10⁴ uninfected cells, lysed, and analyzed by nested PCR to detect viral genome. Curve fit lines were derived from nonlinear regression analysis, and symbols represent mean percentages of wells positive for virus ± the standard error of mean. The dashed line represents 63.2%, from which the frequency of viral genome-positive cells was calculated based on the Poisson distribution. (D) Lung cells from G50.stop-infected mice were harvested and digested with collagenase. Cells were stained with anti-CD19 conjugated to allophycocyanin and anti-B220 conjugated to fluorescein isothiocyanate (BD Pharmingen), sorted by flow cytometry on the MoFlo (Cytomation, Fort Collins, CO), and analyzed by LDPCR. Purity of sorted cells exceeded 94%. (E) Bulk lung cells from G50.stop-infected mice at day 16 postinfection were isolated as described above and assayed by LDPCR as previously described (14, 18). For all experiments, the data shown represent at least three independent experiments with cells pooled from five mice per experimental group.

FIG. 2.

Analysis of γHV68 lytic replication and latency in mice treated with the antiviral drug cidofovir. Mice were given subcutaneous injections of cidofovir (25 mg/kg; Vistide; Gilead Sciences, Foster City, CA) starting at day −1, day 0, and day 2 followed by injections every third day. On day zero, mice were infected with 1,000 PFU γHV68 i.n. (A) Lung tissue was harvested at day 7 postinfection and the titer was determined on NIH 3T12 cells (ATCC CCC 164) as previously described (8). Each data point represents a mouse. (B) Bulk spleen cells were harvested at day 16 postinfection and analyzed for the frequency of cells harboring viral genome as determined by LDPCR as previously described.

FIG. 3.

Determination of the immune response to G50.stop. Lungs were harvested from naïve mice and mice infected with G50.stop and γHV68-CreGFP at day 14 postinfection, collagenase digested, and stained for flow cytometry analysis. Lung cells were stained with anti-mouse CD8, anti-mouse CD4, and anti-CD11a (all from BD Pharmingen) and analyzed on a FACSCalibur (BD Biosciences Immunocytometry Systems, San Jose, CA). Data from representative mice are shown (a total of six mice were individually analyzed for each condition in two independent experiments). (A) Collagenase-digested lung cells were gated on CD8⁺ and CD4⁺ T cells. Numbers represent percentages of lung cells. (B) Plots gated on CD8⁺ T cells. Numbers represent percentages of CD11a^hi CD8⁺ T cells. Among the mice analyzed, there was a statistically significant difference between the percentage of CD11a^hi CD8⁺ T cells between naïve and γHV68-CreGFP-infected mice (P < 0.0001), but not between naïve and G50.stop virus-infected mice (P = 0.58). (C) Plots gated on CD4⁺ T cells. Numbers represent percentages of CD11a^hi CD4⁺ T cells. Among the mice analyzed, there was a statistically significant difference between the percentage of CD11a^hi CD4⁺ T cells between naïve and γHV68-CreGFP-infected mice (P < 0.0001), but not between naïve and G50.stop virus-infected mice (P = 0.20). (D) Plots gated on B cells. The numbers represent percentages of CD69^hi B cells. Among the mice analyzed, there was a statistically significant difference between the percentage of CD69^hi B cells between naïve and γHV68-CreGFP-infected mice (P = 0.0013), but not between naïve and G50.stop virus-infected mice (P = 0.50).

FIG. 4.

Comparison of vaccination with G50.stop and v-cyclin.LacZ. Mice were primed with either medium, 940 PFU v-cyclin.LacZ, or 940 PFU G50.stop i.n. 28 days prior to challenge. Mice were challenged with 100 PFU wild-type γHV68 i.n. (A) Lung tissue was harvested at day 7 postinfection, and the titer was determined on NIH 3T12 cells. Each data point represents a mouse. (B) Bulk spleen cells were harvested at day 16 postinfection and analyzed for cells reactivating virus by an ex vivo limiting dilution reactivation assay as previously described (18, 19). Data are representative of at least two independent experiments. A statistically significant difference was observed between the frequency of viral genome-positive cells in control mice (media) and those immunized with the v-cyclin.LacZ virus (P = 0.0003), but not mice immunized with the G50.stop virus (P = 0.50).