Murine gammaherpesvirus 68 LANA is essential for virus reactivation from splenocytes but not long-term carriage of viral genome

Abstract

ORF73, which encodes the latency-associated nuclear antigen (LANA), is a conserved gamma-2-herpesvirus gene. The murine gammaherpesvirus 68 (MHV68) LANA (mLANA) is critical for efficient virus replication and the establishment of latent infection following intranasal inoculation. To test whether the initial host immune response limits the capacity of mLANA-null virus to traffic to and establish latency in the spleen, we infected type I interferon receptor knockout (IFN-alpha/betaR(-/-)) mice via intranasal inoculation and observed the presence of viral genome-positive splenocytes at day 18 postinfection at approximately 10-fold-lower levels than in the genetically repaired marker rescue-infected mice. However, no mLANA-null virus reactivation from infected IFN-alpha/betaR(-/-) splenocytes was observed. To more thoroughly define a role of mLANA in MHV68 infection, we evaluated the capacity of an mLANA-null virus to establish and maintain infection apart from restriction in the lungs of immunocompetent mice. At day 18 following intraperitoneal infection of C57BL/6 mice, the mLANA-null virus was able to establish a chronic infection in the spleen albeit at a 5-fold-reduced level. However, as in IFN-alpha/betaR(-/-) mice, little or no virus reactivation could be detected from mLANA-null virus-infected splenocytes upon explant. An examination of peritoneal exudate cells (PECs) following intraperitoneal inoculation revealed nearly equivalent frequencies of PECs harboring the mLANA-null virus relative to the marker rescue virus. Furthermore, although significantly compromised, mLANA-null virus reactivation from PECs was detected upon explant. Notably, at later times postinfection, the frequency of mLANA-null genome-positive splenocytes was indistinguishable from that of marker rescue virus-infected animals. Analyses of viral genome-positive splenocytes revealed the absence of viral episomes in mLANA-null infected mice, suggesting that the viral genome is integrated or maintained in a linear state. Thus, these data provide the first evidence that a LANA homolog is directly involved in the formation and/or maintenance of an extrachromosomal viral episome in vivo, which is likely required for the reactivation of MHV68.

FIG. 1.

Intranasal infection of IFN-α/βR^−/− mice with 73.Stop allows greater lytic replication and mLANA-independent seeding of latency in the spleen but not the reactivation of the virus. (A) Either C57BL/6 mice (black symbols) or IFN-α/βR^−/− mice (gray symbols) were infected intranasally with 100 PFU of 73.Stop or 73.MR virus. Lungs were harvested 9 days later, and infectious virus titers were determined by plaque assay. (B) Kaplan-Meier curve of IFN-α/βR^−/− mice infected intranasally with 73.Stop or 73.MR virus. Chi-squared analysis revealed no significant difference in survival between these experimental groups. (C and D) Surviving IFN-α/βR^−/− mice infected with either 73.Stop or 73.MR virus were sacrificed at day 28 postinfection, and the spleen was harvested for analysis. Splenocytes were subjected to limiting-dilution analyses to determine the frequency of cells harboring the viral genome (C) or spontaneously reactivating virus upon explant onto monolayers of mouse embryo fibroblasts (D). rxns, reactions; CPE, cytopathic effect.

FIG. 2.

mLANA is required for efficient reactivation, but not establishment of latency, in PECs and splenocytes following intraperitoneal inoculation of immunocompetent C57BL/6 mice. Groups of three to five female C57BL/6 mice were inoculated intraperitoneally with 1,000 PFU of 73.Stop or 73.MR virus. At day 18 postinfection, PECs (A and C) and splenocytes (B and D) were harvested and assayed for latency and reactivation by limiting-dilution analyses. Results are the means of data from four independent experimental groups, and error bars represent standard deviations between separate groups.

FIG. 3.

B cells account for the majority of mLANA-null virus-infected splenocytes. Splenocytes were harvested from mice at day 18 postinfection and sorted into B-cell and non-B-cell populations by using a commercially available magnetic-bead-based B-cell purification kit (Miltenyi). The bulk and B-cell-positive splenocyte populations were then analyzed by limiting-dilution PCR analysis to determine the frequency of viral genome-positive cells. Purified B cells are shown as black symbols and lines, and the B-cell-depleted splenocyte population is shown as gray symbols and lines.

FIG. 4.

Viral genomes are maintained long-term in mice in the absence of mLANA. C57BL/6 mice were inoculated intraperitoneally with 1,000 PFU of the indicated virus. At 42 days (A) and 6 months (B) postinfection, mice were sacrificed, and splenocytes were assayed by limiting-dilution PCR for the frequency of cells harboring the viral genome.

FIG. 5.

Latency-associated MHV68 transcripts can be detected in mLANA-null virus-infected splenocytes. (A) Semiquantitative RT-PCR analysis of MHV68 latency-associated gene expression in 73.Stop- and 73.MR-infected splenocytes. RNA from individual spleens was reverse transcribed, and dilutions (undiluted or 1:5 or 1:25 dilutions) of the cDNA from each spleen were analyzed by PCR for the indicated genes. Shown are amplifications of products arising from spliced M2 or orf73 transcripts (these RT-PCR products cross the known splice junctions in these viral transcripts), M9, viral DNA polymerase (pol), M1, or the cellular GADPH transcript using RNA harvested from infected mice at days 25 to 28 postinfection. (B) Compiled data from the RT-PCR analyses shown above (A) indicating the number of PCR-positive reactions and the total number of reactions analyzed.

FIG. 6.

mLANA-deficient virus is not impaired in the induction of a strong germinal center response. Shown are compiled data from flow cytometry analyses of the germinal center (GC) response (expressed as a percentage of total B cells that exhibit a germinal center phenotype) in the spleen at 18 days postinfection. Splenocytes from individual mice were stained with anti-CD19, GL7, and anti-CD95.

FIG. 7.

A functional mLANA gene is required for the efficient replication of MHV68 following transfection of viral DNA in permissive fibroblasts. (A) NIH 3T12 fibroblasts were transfected with 0.5 μg of either 73.Stop-BAC or 73.MR-BAC (MR) DNA. At 3, 7, and 9 days posttransfection, supernatants were harvested, and titers of infectious virus produced were determined. The data are representative of at least three replicate experiments. (B) Vero cells were transfected in duplicate as described above, and lysates were harvested at 24, 72, and 96 h. Western blots for lytic MHV68 antigen and β-actin are shown.

FIG. 8.

Digestion-circularization PCR reveals mLANA-dependent episome formation during early latency in the spleen. (A) Diagram of the DC-PCR method. Primers are designed facing away from each other on opposite sides of the TR. After digestion and intramolecular circular formation and ligation, the primers will produce a product during PCR. If the virus is integrated, i.e., does not have fused terminal repeats, only one primer binding site will be contained in the resulting circles, and no product is formed. (B) EcoRI digestion and circularization. Numbers above the gel represent the amounts of digested DNA that went into each ligation reaction mixture. One set of mice was analyzed for 73.MR and 73.Stop infections. The murine acetyl choline receptor (AChR) was a control for digestion and intramolecular circularization. (C) BamHI digestion and circularization. Sixty nanograms of DNA was placed into each ligation reaction mixture. Each pair of lanes (ligase +/−) represents a separate group of mice. Ligase-negative reactions are used as a negative control. DNA from the latently infected HE2 (28) cell line was used as a positive control. Southern blots of the PCR products using probes that span the ligated junction further confirmed the specificity of the PCR. EtBr, ethidium bromide.