Enhancer requirement for murine cytomegalovirus growth and genetic complementation by the human cytomegalovirus enhancer

Abstract

The cytomegalovirus (CMV) enhancer is a highly complex regulatory region containing multiple elements that interact with a variety of host-encoded transcription factors. Many of these sequence elements are conserved among the different species strains of CMV, although the arrangement of the various elements and overall sequence composition of the CMV enhancers differ remarkably. To delineate the importance of this region to a productive infection and to explore the possibility of generating a murine CMV (MCMV) under the control of human CMV (HCMV) genetic elements, the MCMV enhancer was resected and replaced either with nonregulatory sequences or with paralogous sequences from HCMV. The effects of these various deletions and substitutions on viral growth in transfected or infected tissue-culture cells were evaluated. We found that mutations in MCMV that eliminate or substitute for the enhancer with nonregulatory sequences showed a severe deficiency in virus synthesis. This growth defect is effectively complemented by the homologous MCMV enhancer as well as the HCMV enhancer. In the latter case, the chimeric viruses (hybrid MCMV strains) containing the molecularly shuffled human enhancer exhibit infectious kinetics similar to that of parental wild-type and wild-type revertant MCMV. These results also show that open reading frames m124, m124.1, and m125 located within the enhancer region are nonessential for growth of MCMV in cells. Most importantly, we conclude that the enhancer of MCMV is required for optimal infection and that its diverged human counterpart can advantageously replace its role in promoting viral infectivity.

FIG. 1

Construction of enhancerless MCMV BAC genomes. The top line represents the HindIII map of the wild-type (Wt) MCMV genome, with the HindIII K and L regions expanded below to show the region containing the MIE genes (ie1 to ie3). The MCMV BAC plasmids pE1, pSM3dE, and pSM3dE::luc, shown below the wild-type MCMV genome, were generated by homologous recombination in E. coli with pSM3 as the parental MCMV BAC genome as indicated in Materials and Methods. A MunI-HpaI fragment (from +209 to −1421 relative to the ie1/ie3 MCMV transcription start site) in the HindIII L fragment of the wild-type MCMV genome was removed to generate pE1. In pSM3dE, an EspI-NdeI fragment (from nucleotide sequences −48 to −1191 relative to the ie1/ie3 MCMV transcription start site) was deleted in the HindIII L fragment of the wild-type MCMV genome. The MCMV BAC plasmid pSM3dE::luc contains a 770-bp fragment from the luciferase gene replacing nucleotide sequences from −48 to −1191 (relative to the ie1/ie3 MCMV transcription start site) in the HindIII L fragment of the wild-type MCMV genome. The illustration is not drawn to scale.

FIG. 2

Structural analysis of enhancerless MCMV BAC genomes. Ethidium bromide-stained agarose gels of HindIII-digested BAC plasmids pSM3 (1), pE1 (2), pSM3dE (3), and pSM3dE::luc (4) after separation on a 0.5% agarose gel. The HindIII fragment name (11) is indicated to the right of the set of lanes, and the size markers are shown at the left margin. The size of the new HindIII L fragments is indicated with an arrow at the right margin.

FIG. 3

Construction of hMCMV-ES mutants. The top line represents the HindIII map of wild-type MCMV genome, with the HindIII K and L regions expanded below to show the region containing the MIE genes (ie1 to ie3). In the MCMV BAC plasmid pE1, a MunI-HpaI fragment (from nucleotide sequences +209 to −1421 relative to the ie1/ie3 MCMV transcription start site) has been deleted in the HindIII L region of the wild-type MCMV genome. Recombinant viruses MCMVrev and hMCMV-ES were constructed by cotransfection of MCMV BAC plasmid pE1 with pBam25 or pBam25H, respectively, into NIH 3T3 cells. pBam25, a plasmid expressing ie1 and ie3, carries a BamHI fragment containing sequences from 176441 to 187035 of the MCMV genome. pBam25H carries the BamHI fragment (containing sequences from 176441 to 187035 of the MCMV genome) in which sequences from −48 to −1191 (relative to the ie1/ie3 MCMV transcription start site) have been replaced by sequences from −52 to −667 of the HCMV enhancer. The solid and hatched boxes represent the MCMV and HCMV enhancers, respectively. The diagram is not drawn to scale.

FIG. 4

Structural analysis of hMCMV mutants. DNA isolated from NIH 3T3 cells infected with wild-type MCMV (1), MCMVrev (2), hMCMV-ES1 (3), or hMCMV-ES2 (4) was subjected to HindIII digestion and separated on a 0.5% agarose gel. Bands were visualized with ethidium bromide (A) or transferred to nylon filters and hybridized to a ³²P-labeled 340-bp SpeI-SnaBI fragment from the HCMV enhancer (B). The HindIII fragment name (11) is indicated to the left set of lines, and the sizes of the natural and new HindIII L fragments for each virus are shown with an arrow.

FIG. 5

Growth kinetics of hMCMV-ES mutants. Murine NIH 3T3 cells were infected at an MOI of 5 (A) or 0.01 PFU (B) per cell with wild-type (Wt) MCMV (Smith strain), MCMVrev, hMCMV-ES1, or hMCMV-ES2. At the indicated time points (hours) after infection (hpi) supernatants from the infected cultures were harvested, and titers were determined by plaque assay on NIH 3T3 cells. Each data point represents the average and standard deviation from three separate cultures.

FIG. 6

Expression of RNA kinetics of ie1/ie3 transcripts by hMCMV-ES mutants. NIH 3T3 cells were mock infected (lane 1) or infected at an MOI of 0.5 PFU/cell with wild-type MCMV (lanes 2, 6, 10, and 14), hMCMV-ES1 (lanes 3, 7, 11, and 15), hMCMV-ES2 (lanes 4, 8, 12, and 16), and MCMVrev (lanes 5, 9, 13, and 17). Whole-cell RNA was harvested at the indicated times after infection in the presence (lanes 2 to 5) or absence (lanes 1 and 6 to 17) of CH, separated on a formaldehyde-agarose gel, transferred to a nylon membrane, and probed with a 1.6-kb MluI-HindIII fragment from pON401 that was specific for ie1 transcripts. The position of the 2.75-kb transcripts is indicated on the right by an arrow. The positions of the 18S and 28S rRNAs are shown. The blot was then hybridized with a ³²P-labeled GAPDH probe as an internal RNA control. The 1.4-kb GAPDH band detectable in all lanes is shown on the bottom panels.

FIG. 7

Expression kinetics of ie1 protein in hMCMV-ES-infected cells. NIH 3T3 cells were mock infected (lane 1) or infected at an MOI of 0.5 PFU/cell with wild-type MCMV (lanes 2, 6, 10, and 14), hMCMV-ES1 (lanes 3, 7, 11, and 15), hMCMV-ES2 (lanes 4, 8, 12, and 16), and MCMVrev (lanes 4, 9, 13, and 17). At the indicated times after adsorption, samples were lysed, subjected to SDS-PAGE analysis on 8% polyacrylamide gels, and reacted with an ie1 monoclonal antibody. Molecular mass standards (M) appear on the left. The position of the 89-kDa protein is indicated by an arrowhead.