In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4

KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.

In vitro analysis of chimera viruses. The kLANA-C-terminal mLANA fusion proteins were expressed during lytic replication in vitro (Fig. 2b to d). Monoclonal antibody 6A3 recognizes C-terminal mLANA. Monoclonal antibody LN53 recognizes EQEQ epitopes (34) present in the repeat regions comprising the leucine zipper (LZ) and the glutamineglutamate (QE) repeats, just upstream of C-terminal kLANA (Fig. 1c). Since both of these regions are present in kLANA containing the mLANA DBD, both antibodies detected the fusion protein expressed in v-KM.yfp-and v-KM8A10.yfp-infected cells (Fig. 2b, top two panels) by Western blotting. mLANA in v-WT.yfp-infected cells and kLANA in v-kLANA.yfp-infected cells were also detected by monoclonal antibodies (MAbs) 6A3 and LN53, respectively (Fig. 2b, top 2 panels). Similar results were obtained in cells infected with the non-yfp versions of the viruses (Fig. 2c). mORF72, which is part of the same transcript as mORF73 (22), was expressed in all infected cell lysates (Fig. 2b, third panel from top). kLANA and fusion proteins localized to the nucleus of infected cells, with a broad distribution and some regions of more concentrated intensity (Fig. 2d). kLANA disengages from TR DNA during lytic infection, resulting in broad, nuclear distribution (35).
MuHV-4 M11 is a bcl-2 homolog required for efficient establishment of latency (36). M11 is transcribed in the opposite direction of mORF73 (Fig. 2a), and the M11 stop codon overlaps with the 3= end of mORF73. We preserved the M11 stop codon in the chimera constructs. No differences were found in M11 mRNA levels between v-WT.yfp-, v-kLANA.yfp-, and v-KM.yfp-infected cells (Fig. 2e). Thus, phenotypes observed previously with v-kLANA (24) or here with C-terminal swap viruses are not due to altered M11 expression. In addition, M3 and ORF63 are lytic genes that encode a chemokine binding protein (10,37,38) and a tegument protein (39,40), respectively. Neither M3 nor ORF63 mRNA levels were reduced in v-kLANA.yfp or v-KM.yfp compared to v-WT.yfp (Fig. 2e). All viruses grew similarly in vitro (Fig. 2f).
v-KM establishes higher latency levels than v-kLANA. To compare the pathogenesis of the kLANA-C-mLANA MuHV-4 chimera with the wild-type (WT) and the kLANA MuHV-4 chimera, we infected mice intranasally (i.n.) with 10 4 PFU. Typically, at this inoculation dose, lytic virus titers in the lungs peak around day 7. Latent infection in the spleen peaks at around day 14, declining afterwards to very low or undetectable levels.
Lung virus titers were slightly reduced for all chimera viruses compared to wild-type virus (Fig. 3a). This reduction was less than 1 log at day 3 for all recombinants except for v-KM8A10.yfp, which had about 1 log reduction compared to v-WT.yfp (P Ͻ 0.05) (Fig. 3a, left panel). Differences of 0.5 to 1 log between average titers of the chimeric viruses compared to the WT group were also observed at day 7 but did not attain statistical significance (Fig. 3a, right panel).
To quantify latent virus, we performed ex vivo reactivation assays by coculturing total splenocytes with permissive BHK-21 cells. At day 14, reactivating viruses were detected in v-kLANA.yfp-infected mice but, as previously observed, were reduced significantly (P Ͻ 0.01) by nearly 2 log compared to v-WT.yfp infection (Fig. 3b, left  panel). In contrast, v-KM.yfp-infected mice had a mean titer that was intermediate between that of v-kLANA.yfp and that of v-WT.yfp (Fig. 3b, left panel). At day 21, reactivating virus was clearly detectable in the v-KM.yfp infection group, with a mean titer 1 log lower than that of the WT group (P Ͻ 0.05) (Fig. 3b, right panel). In contrast, at this time after infection, v-kLANA.yfp reactivation titers were below or very near the limit of detection of the assay (Fig. 3b, right panel). v-8A10.yfp and v-KM8A10.yfp chimera viruses had no detectable reactivating virus anytime after infection (Fig. 3b). Preformed virus titers assessed in freeze-thawed samples were kLANA-C-mLANA Hybrid Journal of Virology below or at the limit of detection. This confirms that coculture assay titers correspond to reactivation from latency (Fig. 3b). Similar results were obtained in ex vivo reactivation assays at days 14 and 21 after infection with the independent, non-yfp versions of the viruses (Fig. 3c). We determined in parallel the frequency of infection in total splenocytes at day 14 by combining limiting dilution with PCR to detect viral genomes. The v-kLANA.yfp infection group had a 16-fold-lower frequency of infection than that of v-WT.yfp, which is similar to our previous results (24). v-KM.yfp displayed higher frequencies of infection than v-kLANA.yfp, which were closer but still 3.6-fold lower than WT levels ( Fig. 3d and Table 1). Mutants v-8A10.yfp and v-KM8A10.yfp were very reduced, 329-and 400-fold, respectively, compared to the WT group ( Fig. 3d and Table 1). This quantification is independent of the ability of viruses to reactivate ex vivo. Thus, the higher reactivation titers of v-KM.yfp than those of v-kLANA.yfp reflect a higher latency load rather than an in vitro reactivation phenotype.
We also assessed infection in GC B cells at day 14 by flow cytometry (Fig. 4a to c). The mean total number and percentage of GC B cells in the different infectious groups varied from 9.7 ϫ 10 5 to 32.1 ϫ 10 5 and 2.5% to 5.8%, respectively (Fig. 4a, right panels). YFP expression marked infected cells. The mean percentages of GC B cells expressing YFP were 7.6% for v-WT.yfp, 1.1% for v-kLANA.yfp, and 2.3% for v-KM.yfp (Fig. 4b, right panel). The mean percentages of YFP-positive B cells that had a GC phenotype were 85.0%, 68.5%, and 75.8% for v-WT.yfp, v-kLANA.yfp, and v-KM.yfp, respectively (Fig. 4c, right panel). Significantly reduced percentages of YFP-positive GC B cells were observed for the kLANA v-8A10.yfp and v-KM8A10.yfp viruses compared to other groups (P Ͻ 0.01) ( Fig. 4a and b). Taken together, the data indicate that GC B cells were latently infected with v-WT.yfp, v-kLANA.yfp, and v-KM.yfp.
Detection of kLANA-C-terminal mLANA fusion protein in the spleen. To assess expression of the LANA fusion in vivo, we performed immunofluorescence assays of spleen sections of infected mice (Fig. 5a). Control v-WT.yfp-infected spleens had many YFP-positive cells and, as expected since they lack kLANA, no staining with anti-kLANA EQEQ MAb LN53 (Fig. 5a, top panels). v-WT.yfp-and v-KM.yfp-infected spleens had higher frequencies of YFP-positive cells than did v-kLANA.yfp-infected spleens (Fig. 5a). YPF-positive cells from v-KM.yfp and v-kLANA.yfp contained nuclear kLANA dots (Fig. 5a, middle and bottom panels). LANA concentrates to dots at sites of viral episomes, and therefore each dot corresponds to a viral genome (16,21,41). The intensity of YFP expression varied (Fig. 5a, left panels). Infrequently, YFP-negative cells contained kLANA dots (Fig. 5a, arrow in middle panels). This finding is expected, since loss of YFP expression occurs during latent infection, as shown by PCR detection of viral genomes in YFP-negative cells (reference 42 and data not shown). The number of dots per nuclear volume (100 m 3 ) was slightly higher for v-KM.yfp-infected (mean, 12.8; range, 1.6 to 39.2) than for v-kLANA.yfp-infected (mean, 9.7; range, 1.3 to 21.3) cells (P Ͻ 0.05) (Fig. 5b). The number of genomes per fluorescence-activated cell sorter (FACS)-sorted YFP ϩ GC B cell was higher in v-KM.yfp (mean, 99.9; range, 62 to 166.5) than in v-kLANA.yfp (mean, 58.8; range, 12.1 to 82.4) (P Ͻ 0.05) or v-WT.ypf (mean, 66.6; range, 25.1 to 93.9) (statistically not significant) infection groups (Fig. 5c). These data demonstrate that v-KM.yfp and v-kLANA.yfp persist at the WT genome copy number or higher in nuclei of latently infected splenocytes.

DISCUSSION
In this work, we describe a recombinant MuHV-4 encoding a kLANA (amino acids [aa] 1 to 994)-C-terminal mLANA (aa 118 to 314) fusion protein in place of mLANA. The   (19). Given the specific oligomerization mode of the mLANA DBD and the size of the kLANA internal repeat region, ϳ600 residues in length, that is absent from mLANA, steric hindrance and a conflict between these two regions in the fusion protein could occur. It is also possible that these different modes of assembly could lead to different functionalities through binding to distinct partners, which could affect transcription or The latency load of the chimeric v-KM, however, was still lower than that of WT although higher than that of v-kLANA. It is possible that host factors in murine cells may not interact with kLANA regions as efficiently as those from human cells, hampering expansion of cells latently infected with the MuHV-4 expressing the kLANA-C mLANA fusion. It is also possible that kLANA regions could elicit recognition by the mouse immune system, leading to clearance of some virally infected cells.
Both kLANA and the LANA fusion formed LANA dots in the nuclei of infected splenocytes. These dots were similar to mLANA dots that we previously observed in WT-MuHV-4-infected mice (24). Each nuclear dot corresponds to a virus episomal genome, indicating similar genome copy numbers per nucleus for v-kLANA and v-KM. PCR data demonstrated that both v-kLANA and v-KM had similar numbers of genomes per infected GC B cell compared to v-WT. These results are consistent with previous results demonstrating a WT genome copy number in infected splenocyte nuclei for kLANA chimeric virus.
Virus persistence in proliferating, latently infected cells requires episome persistence. Therefore, these data indicate that the kLANA fusion protein is functional for episome maintenance. During episome persistence, LANA tethers viral DNA to mitotic chromosomes to ensure segregation of virus genomes to daughter cell nuclei. Binding to histones is required for LANA attachment to chromosomes. As expected, mutation of the histone binding site of N-terminal kLANA, which abolishes mitotic chromosome association, abolished latency of MuHV-4 expressing full-length kLANA or the fusion protein. This result also highlights the usefulness of the chimera expressing the fusion protein to address in vivo kLANA function.
In conclusion, the recombinant MuHV-4 described here demonstrates that kLANA can functionally accommodate the mLANA DBD, despite its innate differences in DNA binding. Further, it importantly provides an alternative model to investigate non-DBD kLANA regions in vivo. Because this chimeric virus persists at higher latency levels than v-kLANA, it also provides a greater dynamic range for analysis of phenotypes.

MATERIALS AND METHODS
Ethics statement. Animal studies were performed in accordance with the Portuguese official Veterinary Directorate (Portaria 1005/92), European Guideline 86/609/EEC, and Federation of European Laboratory Animal Science Associations guidelines on laboratory animal welfare. Animal experiments were approved by the Portuguese official veterinary department for welfare licensing (protocol AEC_2010_017_PS_Rdt_General) and the IMM Animal Ethics Committee.
Infectivity assays. Viral stocks were prepared by infection of BHK-21 cells (24). Infectious virus titers were determined by plaque assay (suspension assay) in BHK-21 cells. Six-to 8-week-old C57 BL/6J female mice (Charles River) were inoculated intranasally under isoflurane anesthesia with 10 4 PFU in 20 l in phosphate-buffered saline (PBS). Lungs or spleens were harvested at the indicated time points. Lungs were homogenized, freeze-thawed, and titrated in BHK-21 cells. To prepare single-cell suspensions, spleens were mechanically disrupted and filtered through a 100-l cell strainer. Cells were incubated with a hypotonic NH 4 Cl solution for lysis of red blood cells and, after washing, resuspended in 2% fetal bovine serum in PBS for limiting dilution and flow cytometry analysis or resuspended in cell medium for infectivity assays. Reactivating virus was quantified by coculture with BHK-21 cells. Preformed infectious virus was determined by plaque assay in freeze-thawed samples. Plates were incubated for 4 days for plaque assay or 5 days for coculture assay. Cells were fixed with 4% formaldehyde and stained with 0.1% toluidine blue, and viral plaques were counted with a plate microscope.
Western blotting. Cells were washed with PBS and disrupted with ice-cold lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM NaF, 1 mM orthovanadate, 1% Triton X-100, and complete protease inhibitors from Roche). Lysates were cleared by centrifugation. Approximately 1.25 ϫ 10 5 uninfected or infected BHK-21 cell equivalents were loaded per lane for protein detection. Proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibodies.
Flow cytometry. Flow cytometry using single-cell suspensions prepared from spleens was performed as previously described (24). Fluorochrome-conjugated antibodies against CD19, GL-7, and CD95 were used to identify GC B cells. Data were acquired on an LSR Fortessa (BD BioSciences) with DIVA software and analyzed with FlowJo 9.3.2 (Tree Star). YFP-negative (YFP Ϫ ) and YFP-positive (YFP ϩ ) GC B cells were sorted using a BD FACSAria flow cytometer (BD BioSciences). The purity of sorted populations was above 98%.
Frequency of viral-genome-positive cells. The frequency of viral-genome-positive cells was determined by combining limiting dilution with real-time PCR to detect the MHV68 M9 gene as described previously (47).
Quantification of viral genomes. YFP ϩ and YFP Ϫ GC B cells were FACS sorted from individual spleens of infected mice (24). Sorted cells were washed with PBS, resuspended in PBS, diluted 1:3 in lysis buffer (10 mM Tris-HCl [pH 8.3], 3 mM MgCl 2 , 50 mM KCl, 0.45% NP-40, 0.45% Tween 20, and 0.5 mg/ml of proteinase K), and incubated overnight at 37°C. After proteinase K inactivation (95°C for 5 min), samples were assessed in duplicate by qPCR for the MHV68 M9 gene or for the cellular ribosomal protein L8 (Rpl8) gene as described previously (24). PCR products were converted to genome copies by comparison to a standard curve of a plasmid harboring M9 or the rpl8. The number of viral gene copies per cell was obtained by dividing the number of M9 copies by one-half the number of Rpl8 copies.
Immunofluorescence. Cells adhering to coverslips were fixed with 4% paraformaldehyde-PBS for 20 min at room temperature (RT) and incubated with 20 mM glycine in PBS (15 min, RT). Cells were permeabilized (0.1% Triton X-100 -PBS, 5 min), blocked with 10% FBS in PBS (10 min), and incubated with primary antibodies (1 h) followed by incubation with secondary antibodies (30 min), all at RT. Frozen spleen sections were prepared essentially as described previously (48). Spleens were dissected into PBS, fixed with 1% paraformaldehyde, 10 mM sodium periodate, and 75 mM L-lysine in PBS for 24 h at 4°C, and equilibrated in 30% sucrose-PBS for 18 h at 4°C and in 1:1 30% sucrose-optimal cutting temperature