Identification of the rhesus macaque rhadinovirus lytic origin of DNA replication

Abstract

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.

FIG. 1

Schematic representation of the RRV genome and oriLyt. (A) Location of oriLyt mapping between ORF69 and ORF70 contained within cosmid 8. (B) Plasmid construct pRRVO containing the 6.4-kb XbaI fragment. Relative positions of some restriction endonuclease sites, A+T- and G+C-rich regions, and the positions of two putative ORFs are shown. (C) Plasmid constructs used in the transfection-infection assay. The box in the bottom right hand corner defines the symbols used for transcription factor binding sites identified within oriLyt.

FIG. 2

Identification of RRV oriLyt using the transient-transfection infection-replication assay. Total cellular DNA from transfected cells was cleaved with EcoRI and DpnI, separated through a 0.8% agarose gel, transferred to a nylon membrane, and probed with SuperCos (Stratagene). Arrowheads indicate replication products. (A) Autoradiogram of a Southern blot of total telo-RF DNA from cells transfected with RRV cosmids and subsequently infected with RRV. Lanes: 1, cosmid 8; 2, cosmid 9; 3, cosmid 28a; 4, cosmid 44. (B) RRV oriLyt is present within a 6.4-kb XbaI I fragment subcloned from cosmid 8. DNA was treated as for panel A except that blots were probed with pGEM7zf(−). Lanes: 1 and 2, pRRVO-transfected infected telo-RF DNA; 3 and 4, as lanes 1 and 2 except that cells were treated with PFA; 5 and 6, pRRVR1-transfected infected telo-RF DNA.

FIG. 3

RRV oriLyt contains two essential repeat elements. Autoradiograms of Southern blots of DNA from transfected infected cells treated as described in Materials and Methods are shown. Arrowheads indicate replication products. (A) DNA from cells transfected with pRRVL3 (lane 1), pRRVL1 (lane 2), pRRVM1 (lane 3), pRRVL4 (lane 4), or pRRVL2 (lane 5). (B) Replication of a plasmid construct containing both intact G+C- and A+T-rich genomic sequences. Lanes 1 and 2, transfection with plasmid pRRVL5 in mock-infected cells and RRV-infected cells, respectively. (C) The A+T-rich region plus 200 bp of the G+C-rich genomic sequence is required for plasmid replication. Cells were transfected with oriLyt plasmids containing variable amounts of the G+C-rich region or the A+T sequence alone. Lanes: 1, pRRVL7 (nt 112979 to 115016); 2, pRRVL6 (nt 112979 to 114067); 3, pRRVL8 (nt 112979 to 114371).

FIG. 4

Addition of an ORF50 expression construct facilitates origin-dependent DNA replication in the cotransfection replication assay. A Southern blot of the cotransfection replication assay is shown. Telo-RFs were transfected with all four overlapping cosmids plus pRRVO alone (lane 1) or pRRVO and 0.5, 1, or 2 μg of pEXP50 (lanes 2 through 4, respectively). The arrowhead indicates the replication product.

FIG. 5

Comparison of RRV oriLyt and the corresponding region in HHV-8. (A) Alignment of RRV and HHV-8 genomes, showing the genomic regions between ORF69 and vFLIP. (B) Comparison of the 2-kb region from RRV oriLyt and the similar region in HHV-8. The relative positions of two putative ORFs within each region are shown. (C) Schematic representation of the oriLyt region of RRV and the positional homolog of HHV-8. The G+C base pair distribution across each region is shown.