Identification of cis sequences required for lytic DNA replication and packaging of murine gammaherpesvirus 68

Abstract

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.

FIG. 1.

Diagram of the plasmid constructs used in this study to identify the minimal MHV-68 oriLyt. A schematic of the 8.4-kb HindIII-D fragment of the MHV-68 genome is shown on top. Open reading frames, the 100-bp repeat region (striped box), and restriction enzyme sites used in cloning are indicated. Numbers correspond to nucleotide positions on the MHV-68 genome (47). The full-length oriLyt plasmids pMO(+) and pMO(−) span ORFs 67, 68, 69, M10, 72, and M11. Deletion constructs were generated as described in Materials and Methods, with the length of each insert indicated in parentheses. The relative replication efficiencies of the constructs, determined from at least three independent experiments, are summarized on the right, with the normalized replication efficiency of pMO set at 100%. The bold line indicates the minimal oriLyt region mapped in this study. ND, not determined precisely due to the GC-rich nature of the 100-bp repeat; the sizes of these plasmids were estimated by restriction enzyme digestion and agarose gel analysis.

FIG. 2.

Identification of an MHV-68 oriLyt with a replication assay. (A) The 8.4-kb HindIII-D fragment contains a functional oriLyt. pKS, pMO(+), or pMO(−) was transfected into 293T cells, followed by infection with wild-type MHV-68. DNA was harvested and treated with DpnI to digest input plasmid and also with XhoI, which cuts once in each copy of the oriLyt plasmid. The samples were then run on an agarose gel and analyzed by Southern blotting with a radiolabeled pKS probe. HindIII-digested λ DNA served as molecular size markers. (B) Initial analysis of the 8.4-kb oriLyt region. Deletion plasmids pMOΔ1 to -Δ5 were generated and tested in the replication assays as described in A. Arrowhead and upper bracket, DpnI-resistant replicated DNA; lower bracket, DpnI-sensitive input DNA.

FIG. 3.

Mapping the minimal MHV-68 oriLyt. The 5′ deletion plasmids pMOΔ6 to -Δ12, pMOΔ2, and pMOΔ5 (A) or 3′ deletion plasmids pMOΔ13 to -Δ16 and pMOΔ3 (B) were tested in replication assays. DNA was extracted from each sample, digested, and analyzed by Southern blotting with radiolabeled pKS as a probe. Lower bracket, DpnI-sensitive input DNA. Upper bracket, DpnI-resistant replicated DNA.

FIG. 4.

Function of MHV-68 oriLyt during viral reactivation. (A) pMOΔ2 or pKS and an RTA-expressing plasmid, pFlag/MRTA, or pFlag-CMV-2, were cotransfected by electroporation into S11E cells, a murine B-cell line harboring latent MHV-68. At 24 h posttransfection, DNA was prepared from each sample and probed with radiolabeled pKS for Southern blot analysis. Expression of MHV-68 RTA induced latent MHV-68 in S11E cells to reactivate and thus the oriLyt plasmid pMOΔ2 to replicate. (B) The oriLyt deletion constructs pMOΔ5, pMOΔ12, and pMOΔ16 were tested in S11E cells as described for A. Lower bracket, DpnI-sensitive input DNA. Upper bracket, DpnI-resistant replicated DNA.

FIG. 5.

Terminal repeat of MHV-68 contains a functional packaging signal. One and two copies of the MHV-68 terminal repeat were cloned into pMOΔ2 to generate pMOΔ2-1TR and pMOΔ2-2TR, respectively. pMOΔ2, pMOΔ2-1TR, or pMOΔ2-2TR was transfected into 293T cells. At 24 h posttransfection, the samples were infected with MHV-68. At 72 h postinfection, viral particles were harvested from the supernatant, embedded in agarose, lysed in situ, and analyzed by pulsed-field gel electrophoresis and Southern blotting. The membrane was first probed with radiolabeled pKS to reveal the packaged amplicon DNA (A), stripped, and reprobed with a radiolabeled DNA sequence corresponding to the ORF57 promoter on the viral genome (which is absent from the plasmids) to identify the wild-type viral genome being packaged (B).

FIG. 6.

Comparison of oriLyt regions from MHV-68 and HHV-8. The G+C content of the corresponding regions from MHV-68 and HHV-8 is shown. Horizontal lines indicate the relative position of the minimal oriLyt mapped in this study compared to that of HHV-8 oriLyt (5, 24). Window size, 500 bp.