Human cytomegalovirus oriLyt sequence requirements

Abstract

The mechanisms of action and regulation of the human cytomegalovirus (HCMV) lytic-phase DNA replicator, oriLyt, which spans more than 2 kbp in a structurally complex region near the middle of the unique long region (UL), are not understood. Because oriLyt is thought to be essential for promoting initiation of lytic DNA synthesis and may participate in regulating the switch between lytic and latent phases, we undertook a mutational study to better define its sequence requirements. Kanr gene cassette insertions located an oriLyt core region between nucleotides (nt) 91751 and 93299 that is necessary but not sufficient for replicator activity in transient assays. In contrast, insertions into auxiliary regions flanking either side of this core-also required for significant replicator activity-had little effect. To search for essential components within the core region, we made a series of overlapping, roughly 200-bp deletions, and qualitatively and quantitatively assessed the abilities of the resulting constructs to mediate replication. All but one of these deletions produced a significant (i.e., greater than twofold) loss of activity, arguing that sequences across this entire region contribute to replicator function. However, two particularly critical segments separated by a dispensable region, here called essential regions I and II, were identified. Within essential region I, which overlaps the previously identified early transcript SRT, two adjacent but nonoverlapping, roughly 200-bp deletions abolished detectable replication. No single element or motif from the left half of essential region I was found to be essential. Thus, essential region I probably promotes replication through the cooperation of multiple elements. However, four small deletions in the right half of essential region I, which included or lay adjacent to the conserved 31-nt oligopyrimidine tract (referred to as the Y block), abolished or virtually abolished oriLyt activity. Together, these results identify candidate oriLyt sequences within which molecular interactions essential for initiation of oriLyt-mediated DNA synthesis are likely to occur.

FIG. 1

Kan^r cassette insertion mutants. (A) Plasmid constructs. Positions of the Kan^r cassette insertions are plotted in relation to landmark features of oriLyt. The name, insertion coordinate, and relative activity for each mutant are noted at the right. Features located on the oriLyt line above the mutants include the UL59 open reading frame and transcript (16), the 29-bp repeats (triangle), the Y block (hollow box), and the two large, imperfect dyad symmetries A and B (3). The core region is shaded and bound by dotted lines, and the flanking regions within which partially inactivated insertions are represented by a gradient. (B) Transient transfection assay of the Kan^r cassette insertion constructs. The abilities of the constructs described in panel A to mediate DNA replication were tested as described in Materials and Methods (lanes 1 to 14). DpnI-resistant products of replication were detected by Southern blotting; a replica of the resulting autoradiogram is shown. The tested plasmids are indicated at the top of the panel. The marker (lane 16) contains 0.1 ng each of EcoRI-treated plasmids SP54, SP50, and pGEM7Zf(−). Plasmid SP50 (lane 15), the parent to most of the insertions, and the vector pGEM7Zf(−) (lane 17) were transfected in parallel as wild-type and negative standards, respectively, for comparison. (C) Transient assay of the Kan^r cassette insertion constructs from which the insertion was deleted by PstI treatment, leaving a residual PstI linker insertion. Each plasmid was tested in duplicate. The autoradiogram is reproduced here. The deletion constructs, which correspond to the Kan^r insertions described in panel A, are indicated at the top of each lane.

FIG. 2

Overlapping deletions across HCMV oriLyt. (A) A schematic of the PvuII (nt 89796)-to-KpnI (nt 94860) fragment encompassing oriLyt and deletion constructs. The deleted region of each plasmid is indicated by nucleotide coordinates and by a gap in the line corresponding to the position in the oriLyt core. The open box in the line of pYZ20+1r represents the reiterated dyad sequence (2). Relative replication efficiencies are given at the right; ND, none detected. (B) Quantitative replication assay. The indicated test plasmids were cotransfected with pSP50 or pSP54 and pGEM-7Zf(−) as described in Materials and Methods. An autoradiogram of the resulting Southern blot is reproduced here. Deletion mutants pSP90, pSP72-24, pLH13Δ, pLH34Δ, and pLH50Δ were cotransfected with pSP54 as the positive internal standard. The other deletion mutants were pSP54 derived, and SP50 was used for the internal wild-type comparison. The pSP68 sample was treated with DpnI plus EcoRI and HindIII because pSP68 lacks an EcoRI site, and thus the pSP50 internal standard migrated to the position of pGEM-7Zf(−). (C) Qualitative assay. The indicated test plasmids were assayed for replication competence by transient transfection as described in Materials and Methods. A representative autoradiogram is reproduced here; only the region of the autoradiogram containing replicated (rep’d) signals is shown.

FIG. 3

Replicator activities of insertion and deletion mutants relative to those of the wild type. Exterior deletions that impinge upon the oriLyt region (thick grey bar) progressively reduce activity (3, 22). The positions and relative replicator activities of insertions (closed triangles) and deletions (thin grey bars) are plotted. The trough in the activity plot defines the core region (cross-hatched rectangle). Essential regions I and II, defined by deletions that completely abrogated replicator activity (black rectangles I and II), and the intervening deletable segment (open rectangle D) are indicated.

FIG. 4

Mutations in essential region I. (A) A schematic of the mutations in essential region I. Essential region I is enlarged directly below a schematic of the oriLyt region highlighting landmark features. For each plasmid, the deleted sequence is indicated by nucleotide coordinates and by a gap in the line. Relative replication efficiencies estimated in the quantitative replication assay are noted at the right; ND, none detected. (B) Quantitative replication assay of the small deletions. For each test plasmid, pSP50 and pGEM-7Zf(−) were used as wild-type and negative internal standards, respectively. The relative replication efficiencies of each plasmid were measured as described in Materials and Methods and are indicated at the right of in panel A. Samples for lanes 14 to 17 were from a transfection experiment and blot separate from those for lanes 1 to 13.