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. 2011 Feb;85(3):1330-9.
doi: 10.1128/JVI.01411-10. Epub 2010 Nov 17.

Cloning of the Epstein-Barr virus-related rhesus lymphocryptovirus as a bacterial artificial chromosome: a loss-of-function mutation of the rhBARF1 immune evasion gene

Makoto Ohashi  1 , Nina Orlova, Carol Quink, Fred Wang
Affiliations

Cloning of the Epstein-Barr virus-related rhesus lymphocryptovirus as a bacterial artificial chromosome: a loss-of-function mutation of the rhBARF1 immune evasion gene

Makoto Ohashi et al. J Virol. 2011 Feb.

Abstract

Rhesus macaques are naturally infected with a gammaherpesvirus which is in the same lymphocryptovirus (LCV) genus as and closely related to Epstein-Barr virus (EBV). The rhesus macaque LCV (rhLCV) contains a repertoire of genes identical to that of EBV, and experimental rhLCV infection of naive rhesus macaques accurately models acute and persistent EBV infection of humans. We cloned the LCL8664 rhLCV strain as a bacterial artificial chromosome to create recombinant rhLCV for investigation in this animal model system. A recombinant rhLCV (clone 16 rhLCV) carrying a mutation in the putative immune evasion gene rhBARF1 was created along with a rescued wild-type (rWT) rhLCV in which the rhBARF1 open reading frame (ORF) was repaired. The rWT rhLCV molecular clone demonstrated viral replication and B-cell immortalization properties comparable to those of the naturally derived LCL8664 rhLCV. Qualitatively, clone 16 rhLCV carrying a mutated rhBARF1 was competent for viral replication and B-cell immortalization, but quantitative assays showed that clone 16 rhLCV immortalized B cells less efficiently than LCL8664 and rWT rhLCV. Functional studies showed that rhBARF1 could block CSF-1 cytokine signaling as well as EBV BARF1, whereas the truncated rhBARF1 from clone 16 rhLCV was a loss-of-function mutant. These recombinant rhLCV can be used in the rhesus macaque animal model system to better understand how a putative viral immune evasion gene contributes to the pathogenesis of acute and persistent EBV infection. The development of a genetic system for making recombinant rhLCV constitutes a major advance in the study of EBV pathogenesis in the rhesus macaque animal model.

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Figures

FIG. 1.
FIG. 1.
Schema of the rhLCV genome and restriction fragment analysis of the clone 16 rhLCV BAC. (A) BamHI restriction map of the LCL8664 rhLCV genome. The relative size and position of each rhLCV BamHI DNA fragment are shown in the LCL8664 rhLCV episome. BAC vector sequences were inserted into the BamHI-B fragment by homologous recombination in LCL8664 cells in order to recover the clone 16 rhLCV BAC. The terminal repeats (TR) and major internal repeat (IR1) are represented by boxes. The major transcriptional units for the latent infection genes are represented by the solid arrows and shown for orientation. (B) Restriction digestion analysis of clone 16 rhLCV BAC. Clone 16 rhLCV BAC DNA digested with BamHI, EcoRI, or HindIII and separated by gel electrophoresis was visualized by ethidium bromide staining and UV illumination (left). The predicted sizes of the BamHI, EcoRI, and HindIII fragments in the LCL8664 rhLCV genome and clone 16 rhLCV BAC are shown in Table S1 in the supplemental material. The relative migration of high-molecular-weight markers (HMW) and low-molecular-weight markers (LMW) is shown. BamHI-digested clone 16 rhLCV BAC DNA was transferred to a nylon membrane and hybridized with a pool of radiolabeled oligonucleotide probes in order to identify the BamHI-A, -E, -I, -K, -M, -Q, -U, -X, -a, and -e DNA fragments (right).
FIG. 2.
FIG. 2.
BRLF1-dependent lytic replication of clone 16 rhLCV BAC in C33A cells. Two individual colonies of C33A cells stably carrying clone 16 rhLCV BAC were induced for lytic viral replication, and viral DNA separated by Gardella gel electrophoresis of induced cells was detected on Southern blots. Cells were induced for lytic replication by various combinations of adenoviruses expressing BZLF1 (AdBZLF1) or BRLF1 (AdBRLF1) and chemical treatment with phorbol ester and butyrate (TPA/But). Migration of the 23-kb DNA marker is shown on the left.
FIG. 3.
FIG. 3.
Recovery of BAC-derived clone 16 rhLCV in an LCL, Cre-mediated excision of BAC vector sequences from the viral episome, and predicted amino acid sequence for the truncated rhBARF1 ORF. (A) Schematic of the EcoRI-G rhLCV DNA fragment containing the rhBARF1 ORF in LCL8664 rhLCV, BAC-derived clone 16 rhLCV before (pre-cre) and after (post-cre) Cre-mediated excision, and post-Cre, BAC-derived rWT rhLCV constructed by repairing the rhBARF1 ORF from clone 16 rhLCV BAC. EcoRI digestion sites (E) and locations of PCR primers (arrowheads) are shown. (B) Confirmation of recombinant rhLCV DNA in an LCL infected with clone 16 rhLCV (pre-Cre). Genomic DNA from an LCL immortalized with LCL8664 rhLCV or pre-Cre clone 16 rhLCV was digested with EcoRI, and Southern blots were hybridized with a radiolabeled EcoRI-G rhLCV DNA fragment to detect restriction fragment polymorphisms associated with the insertion of the BAC vector in the viral DNA. (C) PCR confirmation of successful Cre-mediated excision of the BAC vector in the rhLCV clone 16 LCL. Genomic DNA from LCL immortalized with LCL8664 rhLCV, pre-Cre clone 16 rhLCV, or post-Cre clone 16 rhLCV was amplified with the primers shown in panel A. PCR products were hybridized with a radiolabeled oligonucleotide probe derived from the rhBARF1 ORF (top) or the loxP sequence (bottom). (D) Effects of the loxP scar sequence on the rhBARF1 amino acid sequence from clone 16 rhLCV. PCR amplified DNA from LCL infected with clone 16 rhLCV was sequenced, and amino acid sequences from residues 140 to 160 are shown. The loxP scar sequence results in an in-frame insertion of five new amino acids (italicized in the bottom sequence) and a premature termination codon (*). rhBARF1 amino acid residues highly conserved in CSF-1R and other c-Fms family members are underlined.
FIG. 4.
FIG. 4.
Comparison of B-cell-transforming activity by LCL8664, rWT, and clone 16 rhLCV. (A) Transforming activity in multiple virus preparations of LCL8664, rWT, and clone 16 rhLCV. Different virus-producing LCL infected with LCL8664 (lines A, B, and C), rWTrhLCV (lines A and B), and clone 16 rhLCV (lines A, B, and C) were used to make multiple virus preparations. Transforming activity (DNA/TU) was evaluated by calculating the number of relative viral DNA units required per transforming unit (TU). Virus preparations were PCR amplified together in two separate experiments, and the relative DNA units were normalized so that the transforming activity of the naturally occurring LCL8664 rhLCV was 10 DNA units/TU. (B) The efficiency of transforming activity (DNA/TU) for all virus preparations in experiments 1 and 2 is graphed. The overall average DNA/TU for LCL8664 rhLCV was 10.0 (n = 10), 4.6 for rWT rhLCV (n = 9), and 90.3 for clone 16 rhLCV (n = 13). P values (t test) less than 0.05 are shown. (C) rhLMP2A mRNA expression in LCL8664-, rWT-, and clone 16 rhLCV-infected LCL. Total RNA from LCL was reverse transcribed with gene-specific primers, and 10-fold serial dilutions (1 to 10−4) of cDNA were PCR amplified for rhLMP2A or GAPDH.
FIG. 5.
FIG. 5.
Wild-type rhBARF1, but not the truncated rhBARF1, is readily secreted and capable of blocking CSF-1-dependent cell growth. (A) Expression of recombinant BARF1 homologues from EBV (BARF1), rWT rhLCV (rWT rhBARF1), and clone 16 rhLCV (clone 16 rhBARF1). ORFs were tagged with a carboxy-terminal Flag epitope, and expression vectors, or a pSG5-Flag vector control (Ctrl), were transiently transfected into 293 cells. Protein expression in cell lysates or cell-free supernatants was detected by immunoblotting with an anti-Flag monoclonal antibody. Relative migration of 31- and 21.5-kDa molecular mass markers is shown. (B) Inhibition of CSF-1-dependent cell growth by wild-type BARF1 and rhBARF1. BARF1 and rhLCV proteins were immunoprecipitated with an anti-Flag antibody from cell supernatants or cell lysates of transfected 293 cells or recombinant vaccinia virus-infected BSC40 cells. Decreasing amounts of recombinant protein were added to duplicate microwells of CSF-1-dependent BAC1 2F5 cells supplemented with 9 ng/ml of recombinant CSF1, and cell growth was measured by a colorimetric assay after 3 days. The background value from BAC1 2F5 cells without CSF-1 was subtracted from all values, and maximal growth was derived from BAC1 2F5 cells supplemented with CSF-1 only. The growth inhibition after addition of BARF1 (closed diamonds), rWT rhBARF1 (closed circles; from supernatant and cell lysate), clone 16 rhBARF1 (closed squares), rhBZLF1 (open squares), and rhBVDF1 (open diamonds) is plotted for each dose of recombinant protein. Proteins affinity purified from cell supernatants are shown with a solid line, and proteins affinity purified from cell lysates are shown with a dotted line.
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