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. 2002 Jan;76(1):313-26.
doi: 10.1128/jvi.76.1.313-326.2002.

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication

Jeffery L Meier  1 , Michael J Keller, James J McCoy
Affiliations

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication

Jeffery L Meier et al. J Virol. 2002 Jan.

Abstract

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer's orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at -300 or -345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

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Figures

FIG. 1.
FIG. 1.
Effects on viral DNA replication of successively larger 5′-subsegmental distal enhancer deletions. (A) Schematic diagram of deleted distal enhancer segments in recombinant HCMVs rΔ-300/-579, rΔ-347/-579, and rΔ-521/-579. Base positions of deletions and PstI sites are depicted relative to the +1 RNA start-site of the MIE promoter. Predicted sizes of PstI RFLPs are shown. MIE, MIE transcription unit; ENH, enhancer; MOD, modulator; UL128, putative UL128 gene. (B) Southern blot analyses of PstI RFLPs of WT, rΔ-300/-579, rΔ-347/-579, rΔ-521/-579, and rΔMSVgpt. rΔ-300/-579, rΔ-347/-579, and rΔ-521/-579 were derived from rΔMSVgpt. Probe coordinates are given in base positions relative to the RNA start-site of the MIE promoter. (C) Abundances of WT, rΔ-347/-579, and rΔ-521/-579 genomes in HFF cells at MOI of 1.0 and 0.001. Infections were performed in parallel with equivalent input viral titers (see Materials and Methods). On the indicated day p.i. (DPI), infected-cell DNA was isolated, digested with HindIII, fractionated by gel electrophoresis, and subjected to Southern blot analysis. The 32P-labeled probe hybridizes to HCMV genomic fragments containing either the terminal repeat-long (TRL) or the internal repeat-long (IRL) region (23). The IRL is fused (Fused End) to the short genome segment, which is contained in the 17.2- and 13-kb fragments. The TRL is not fused (Free End) to the short genome segment and is contained in the 9.7-kb fragment. The blot was stripped and rehybridized to a λ-specific, 32P-labeled probe for detection of the lambda DNA internal control (Control). (D) Abundances of WT, rΔ-347/-579, and rΔ-300/-579 genomes in HFF cells at MOI of 1.0 and 0.001. The analysis was performed as described for Fig. 1C.
FIG. 1.
FIG. 1.
Effects on viral DNA replication of successively larger 5′-subsegmental distal enhancer deletions. (A) Schematic diagram of deleted distal enhancer segments in recombinant HCMVs rΔ-300/-579, rΔ-347/-579, and rΔ-521/-579. Base positions of deletions and PstI sites are depicted relative to the +1 RNA start-site of the MIE promoter. Predicted sizes of PstI RFLPs are shown. MIE, MIE transcription unit; ENH, enhancer; MOD, modulator; UL128, putative UL128 gene. (B) Southern blot analyses of PstI RFLPs of WT, rΔ-300/-579, rΔ-347/-579, rΔ-521/-579, and rΔMSVgpt. rΔ-300/-579, rΔ-347/-579, and rΔ-521/-579 were derived from rΔMSVgpt. Probe coordinates are given in base positions relative to the RNA start-site of the MIE promoter. (C) Abundances of WT, rΔ-347/-579, and rΔ-521/-579 genomes in HFF cells at MOI of 1.0 and 0.001. Infections were performed in parallel with equivalent input viral titers (see Materials and Methods). On the indicated day p.i. (DPI), infected-cell DNA was isolated, digested with HindIII, fractionated by gel electrophoresis, and subjected to Southern blot analysis. The 32P-labeled probe hybridizes to HCMV genomic fragments containing either the terminal repeat-long (TRL) or the internal repeat-long (IRL) region (23). The IRL is fused (Fused End) to the short genome segment, which is contained in the 17.2- and 13-kb fragments. The TRL is not fused (Free End) to the short genome segment and is contained in the 9.7-kb fragment. The blot was stripped and rehybridized to a λ-specific, 32P-labeled probe for detection of the lambda DNA internal control (Control). (D) Abundances of WT, rΔ-347/-579, and rΔ-300/-579 genomes in HFF cells at MOI of 1.0 and 0.001. The analysis was performed as described for Fig. 1C.
FIG. 2.
FIG. 2.
Multiple distal enhancer parts are involved in augmenting viral DNA replication. (A) Schematic diagram of deleted distal enhancer segments in HCMVs rΔ-347/-579 and rΔ-300/-347. Base positions of deletions and PstI sites are depicted relative to the RNA start site of the MIE promoter. Predicted sizes of PstI RFLPs are shown. (B) Southern blot analyses of PstI RFLPs of WT, rΔ-347/-579, rΔ-300/-347, and rΔMSVgpt. Probe coordinates are given relative to the RNA start site of the MIE promoter. (C) Abundances of WT, rΔ-300/-347, rΔ-347/-579, and rΔ-300/-579 genomes in HFF cells at an MOI of 1.0 on days 2 and 3 p.i. (DPI 2 and 3). (D) Abundances of WT, rΔ-300/-347, rΔ-347/-579, and rΔ-300/-579 genomes in HFF cells at an MOI of 0.005 on days 6 and 8 p.i.. Infections for which results are shown in panels C and D were performed in parallel, using methods described in the legend to Fig 1C.
FIG. 2.
FIG. 2.
Multiple distal enhancer parts are involved in augmenting viral DNA replication. (A) Schematic diagram of deleted distal enhancer segments in HCMVs rΔ-347/-579 and rΔ-300/-347. Base positions of deletions and PstI sites are depicted relative to the RNA start site of the MIE promoter. Predicted sizes of PstI RFLPs are shown. (B) Southern blot analyses of PstI RFLPs of WT, rΔ-347/-579, rΔ-300/-347, and rΔMSVgpt. Probe coordinates are given relative to the RNA start site of the MIE promoter. (C) Abundances of WT, rΔ-300/-347, rΔ-347/-579, and rΔ-300/-579 genomes in HFF cells at an MOI of 1.0 on days 2 and 3 p.i. (DPI 2 and 3). (D) Abundances of WT, rΔ-300/-347, rΔ-347/-579, and rΔ-300/-579 genomes in HFF cells at an MOI of 0.005 on days 6 and 8 p.i.. Infections for which results are shown in panels C and D were performed in parallel, using methods described in the legend to Fig 1C.
FIG. 3.
FIG. 3.
Multiple distal enhancer parts are involved in augmenting MIE promoter-dependent transcription. Relative quantitation of WT, rΔ-300/-347, rΔ-347/-579, and rΔ-300/-579 MIE RNAs at 6 h p.i. in HFF cells was performed for quadruplicate samples by multiplex real-time RT-PCR, using the CT method and validation experiments according to the manufacturer’s specifications. Viral MIE RNA abundance was normalized to cellular 18S RNA (MIE RNAN) and expressed relative (REL) to that of WT at an MOI of 1.0 (A) or 0.005 (B). Shown are means and standard deviations. Parallel determinations of CT values of corresponding samples lacking RT and of mock-infected samples revealed no appreciable difference from the baseline.
FIG. 4.
FIG. 4.
Stop codons inserted into the distal enhancer do not alter viral DNA replication. (A) Schematic diagram of HCMVs r-300.A and rΔ-300.AS. (B) Southern blot analyses of PstI RFLPs of WT, r-300.A, rΔ-300.AS, and rΔMSVgpt. Probe coordinates relative to the RNA start site of the MIE promoter and predicted sizes of PstI RFLPs are given. (C) Abundances of WT, r-300.A, rΔ-300.AS, and rΔ-300/-579 genomes in HFF cells at MOI of 1.0 and 0.001 on days 2 and 7 p.i. (DPI 2 and 7). Infections were performed in parallel by methods described in the legend to Fig. 1C.
FIG. 5.
FIG. 5.
An inverted distal enhancer augments viral DNA replication. (A) Schematic diagram of WT and r-579/-300. Base positions of PstI, NdeI, and NcoI sites and probe (solid bar) coordinates are given relative to the RNA start site of the MIE promoter. INV, inverted distal enhancer. (B) Southern blot analyses of PstI and combined NdeI and NcoI RFLPs of WT, r-579/-300, and rΔMSVgpt. Predicted sizes of RFLPs are shown. (C) Abundances of WT, r-579/-300, and rΔ-300/-579 genomes in HFF cells at an MOI of 1.0 on days 2 and 3 p.i. (DPI 2 and 3). (D) DNA replication rates of WT, r-579/-300, and rΔ-300/-579 genomes in HFF cells at an MOI of 0.005 on days 6 and 7 p.i. Infections for which results are shown in panels C and D were performed in parallel by methods described in the legend to Fig. 1C. (E) Abundances of WT, r-579/-300a, r-579/-300b, and rΔ-300/-579 genomes in HFF cells at an MOI of 0.005 on day 7 p.i. Viruses r-579/-300a and r-579/-300b were derived from independent transfection-recombination procedures. The abundances of viral genomes at 4 h, 2 days, and 3 days p.i. at an MOI of 1 were equivalent for all four viruses (data not shown). Analysis was performed as described in the legend to Fig. 1C.
FIG. 6.
FIG. 6.
An inverted distal enhancer augments MIE promoter-dependent transcription. Relative quantitation of WT, r-579/-300, and rΔ-300/-579 MIE RNAs at 6 h p.i. in HFF cells was performed for quadruplicate samples by multiplex real-time RT-PCR, using the CT method and validation experiments according to the manufacturer’s specifications. The standard curve method was also used in parallel to validate results. Comparative input viral titers are shown in Fig. 5C and D. Viral MIE RNA abundance was normalized to that of cellular 18S RNA (MIE RNAN) and expressed relative (REL) to WT at an MOI of 1.0 (A) or 0.005 (B). Shown are means and standard deviations. Parallel determinations of CT values of corresponding samples lacking RT and of mock-infected samples revealed no appreciable difference from the baseline.
FIG. 7.
FIG. 7.
UV-inactivated HCMV virions applied at a high viral-particle/cell ratio greatly increase the ability of rΔ-300/-579 to produce plaques at low MOI. Purified WTUV (3 to 5 PFU/cell prior to UV inactivation) was applied at the time of infection of HFF cells with WT or rΔ-300/-579 at an MOI of 0.001. After viral absorption, cells were washed and covered with agarose. Viral plaques were visualized by light microscopy and enumerated. Mean plaque numbers and SDs were derived from triplicate experiments performed in parallel.
FIG. 8.
FIG. 8.
Augmentation of rΔ-300/-579 MIE promoter-dependent transcription by rΔ-300/-1108EgfpUV at a high viral-particle/cell ratio. HFF cells were infected with rΔ-300/-579 at an MOI of 0.005, and rΔ-300/-1108EgfpUV (MOI of 3 prior to UV inactivation) was added concomitantly to or omitted from the infection. Relative quantitation of viral MIE and cellular 18S RNAs was performed on quadruplicate samples by multiplex real-time RT-PCR, using the CT method and validation experiments according to the manufacturer’s specifications. Viral MIE RNA abundance was normalized to that of 18S RNA (MIE RNAN) and expressed relative (REL) to that of rΔ-300/-579 in the absence of rΔ-300/-1108EgfpUV. Shown are means and standard deviations. Parallel determinations of CT values of corresponding samples lacking RT and of mock-infected samples revealed no appreciable difference from the baseline.
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