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. 1999 Jun;73(6):5043-55.
doi: 10.1128/JVI.73.6.5043-5055.1999.

In vivo replication of recombinant murine cytomegalovirus driven by the paralogous major immediate-early promoter-enhancer of human cytomegalovirus

N K Grzimek  1 , J Podlech, H P Steffens, R Holtappels, S Schmalz, M J Reddehase
Affiliations

In vivo replication of recombinant murine cytomegalovirus driven by the paralogous major immediate-early promoter-enhancer of human cytomegalovirus

N K Grzimek et al. J Virol. 1999 Jun.

Abstract

Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.

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Figures

FIG. 1
FIG. 1
Construction and verification of recombinant viruses. All map positions are given relative to the 5′ start site (counted as +1) of the ie1/3 transcription unit of mCMV, and illustrations are drawn to scale. (A) Map of plasmid constructs for homologous recombination. The HindIII physical map of the mCMV Smith strain genome is shown at the top. For the construction of the mCMV MIEPE deletion plasmid pAMB25ΔMIEPE, plasmid pAMB25 was digested with MluI and MunI and surplus deletions were restored by PCR, resulting in a final deletion of 1,267 bp (ΔMIEPE). Arrows indicate the orientations of ie1/3 and ie2 transcription. Plasmid phCMVMIEPE-gpt.lacZ was generated by insertion of the hCMV enhancer and core promoter (solid box, representing hCMV nucleotides −14 to −601 relative to the start site of the hCMV ie1-ie2 transcription unit) and of a gpt.lacZ reporter gene cassette flanked by loxP sites (asterisks). (B) MIEPE swap mutants. Maps are shown on the left and the corresponding HindIII cleavage analysis is shown on the right. (Left) Expanded HindIII fragments K and L of the mCMV Smith strain genome are shown on the top, illustrating the location of the MIEPE and flanking ie sequences within the authentic 7.1-kbp L fragment (map corresponding to lane 1). Replacement of the mCMV MIEPE by the hCMV MIEPE (solid box) and insertion of a gpt.lacZ reporter gene cassette flanked by loxP sites (asterisks) created novel HindIII fragments of 0.8, 2.84, and 8.26 kbp in the genome of recombinant virus mCMVhMIEPE-gpt.lacZ (map corresponding to lane 2). For the generation of recombinant virus mCMVhMIEPE, the gpt-lacZ cassette was removed from mCMVhMIEPE-gpt.lacZ via Cre recombinase-mediated loxP-specific recombination, leaving a single loxP site (asterisk) and generating a shortened HindIII L fragment of 6.51 kbp (map corresponding to lane 3). (Right) Purified virion DNA was subjected to cleavage by HindIII, and fragments were analyzed by agarose gel electrophoresis, Southern blot, and hybridization with MIEPE type-specific γ-32P-end-labeled oligonucleotide probes. Lanes: M, indicated size markers; 1, DNA of parental virus mCMVΔorf152; 2, DNA of mCMVhMIEPE-gpt.lacZ; 3, DNA of mCMVhMIEPE. Left panel, ethidium bromide-stained gel; center panel, autoradiograph obtained after hybridization of the Southern blot with the 30-bp probe mE-oligo-P; right panel, autoradiograph obtained after stripping of the same filter followed by hybridization with the 30-bp oligonucleotide probe hE-oligo-P. See Fig. 3 for the map locations of the two probes.
FIG. 2
FIG. 2
Determination of the genome-to-infectivity ratios for prototype mCMV Smith, parental mCMVΔorf152, and MIEPE swap mutant mCMVhMIEPE. Purified virion DNA, corresponding to known infectivity measured as noncentrifugal PFU in fibroblast monolayer cultures, was serially diluted in independent duplicates and was subjected to PCR, amplifying a 363-bp fragment of ie1 gene exon 4. Defined numbers of plasmid pIE111, encompassing gene ie1, were subjected to PCR as a standard. (Top) Autoradiograph of a Southern dot blot obtained after hybridization with a γ-32P-end-labeled internal oligonucleotide probe. (Bottom) Computed phosphorimaging results of the same blot. Log-log plots of radioactivity (means of duplicates) measured as phosphostimulated luminescence (PSL) units (ordinate) versus the DNA dilutions expressed in terms of PFU (abscissa) are shown. The upper rule relates the PFU to the number of pIE111 plasmids in the standard. The calculation from the linear portions of the graphs is demonstrated for 1 PFU corresponding to 500 molecules of pIE111 standard template. Thus, the genome-to-PFU ratio is 500:1 in this example.
FIG. 3
FIG. 3
Map of MIEPE type-specific hybridization probes. (Top) Structural organization of the authentic MIEPE of mCMV (mMIEPE), essentially based on data by Dorsch-Häsler et al. (11). Map positions refer to the 5′ start site (counted as +1) of the ie1/3 transcription unit. The map location of oligonucleotide probe mE-oligo-P used for Southern blot hybridization (see Fig. 1B) is indicated. The red bar represents the polynucleotide probe mMIEPE-P used for mCMV MIEPE-specific red staining in the two-color ISH. (Bottom) Structural organization of the chimeric region in recombinant virus mCMVhMIEPE. Sequences of the inserted hCMV core promoter and enhancer (hMIEPE), representing positions −14 to −601 in hCMV, are highlighted by yellow shading. Map positions refer to the 5′ start site (counted as +1) of the mCMV ie1/3 transcription unit. The map location of oligonucleotide probe hE-oligo-P (see Fig. 1B) is indicated. The long black bar represents the polynucleotide probe hMIEPE-P used for hCMV MIEPE-specific black staining in the two-color ISH. The two maps are drawn to scale. Consensus sequences within 18- and 19-bp direct repeats, encompassing NF-κB and CREB/ATF binding sites, are marked by blue and green coloring, respectively.
FIG. 4
FIG. 4
Two-color MIEPE type-specific ISH in liver tissue sections. After immunoablative treatment, groups of BALB/c mice were infected intravenously with either mCMV or recombinant virus mCMVhMIEPE or were coinfected with both viruses. Histological analysis was performed on day 9 after infection. Three serial sections, sharing a central vein as a landmark, were selected for hybridization. The first section of each series was hybridized with a polynucleotide probe specific for the MIEPE of mCMV (mMIEPE-P; red staining), the second was hybridized with a polynucleotide probe specific for the MIEPE of hCMV (hMIEPE-P; black staining), and the third was hybridized with a mixture of both probes. See Fig. 3 for the map locations of the two probes. Results are documented for all nine possible combinations. Counterstaining was performed with hematoxylin. The bar in the bottom right panel represents 50 μm.
FIG. 5
FIG. 5
Replication of mCMV and mCMVhMIEPE in mouse liver parenchyma after coinfection. Mutually exclusive, independent infection of hepatocytes by the two viruses is documented by a MIEPE type-specific, single-cell two-color ISH analysis. Three serial, directly neighboring 2-μm sections were hybridized with probe mMIEPE-P, staining mCMV DNA in red (A); with probe hMIEPE-P, staining mCMVhMIEPE DNA in black (B); and with both probes (C). A central vein in the upper right corner serves as a landmark. The single arrow tracks an mCMV-infected hepatocyte through the section series. Note the particularly prominent intranuclear inclusion body stained by mMIEPE-P but not by hMIEPE-P. Likewise, the twin arrows mark a cell couple that is infected by the MIEPE swap recombinant virus mCMVhMIEPE, as we can conclude from the black staining with hMIEPE-P. Counterstaining was performed with hematoxylin. The bar in panel C represents 50 μm.
FIG. 6
FIG. 6
Quantitative analysis of virus growth in coinfected tissues. (A) Illustration of area morphometric analysis. MIEPE type-specific two-color ISH with hybridization probes mMIEPE-P (red staining) and hMIEPE-P (black staining) was employed to distinguish infectious foci caused by the viruses mCMV and mCMVhMIEPE, respectively, in liver parenchyma on day 9 after coinfection. Foci were circumscribed by a virtual line, and the area enclosed by the resulting polygon was calculated by using area morphometry software. The bar represents 40 μm. (B) Kinetics of virus growth in liver parenchyma. Results of area morphometric analyses performed on days 6 and 9 after coinfection are documented for representative 30-mm2 areas of tissue compiled from three livers per time point. Histograms represent the number of foci (ordinate) per classified focus size (abscissa) with size classes of 4,000 μm2. A threshold was set at 2,000 μm2, which represents ca. four hepatocytes. N, total number of foci within a 30-mm2 area of tissue. Arrows point to the size class of the particular foci shown in panel A. (C) Comparative quantitation of virus replication in the liver and at extrahepatic sites. The numbers of cells infected with mCMV (red) and mCMVhMIEPE (black) were determined on day 9 after coinfection for representative 10-mm2 areas of tissue sections derived from the organs indicated. In the case of adrenal glands, infected cells were counted for 2-mm2 areas, and the number was extrapolated to 10 mm2, whereas infected cells were counted for 10-mm2 areas in the case of the other organs. Shown are linked data pairs for mouse 1 through mouse 5 in order to document the variance between individual mice.
FIG. 7
FIG. 7
Tissue distribution and cell-type tropism of MIEPE swap mutant mCMVhMIEPE. Immunohistological analysis specific for the IE1 protein was performed on day 9 after infection with mCMVhMIEPE. (A1 to A3) Infection of liver parenchyma. A1, overview demonstrating advanced foci of infection; A2, enlargement of the portion of A1 indicated by the arrow, demonstrating the infection’s plaque-like character with a necrotic center. Infected cells were mostly hepatocytes, but endothelial cells (arrowhead) and a few Kupffer cells (not visible) were also present. A3, detail showing condensation of the IE1 protein in the intranuclear inclusion body of infected hepatocytes. (B1 to B2) Infection of perifollicular stromal cells in the spleen. B1, overview; B2, detail of the portion of B1 indicated by the arrow. f, remnants of follicles. (C) Infection of bone marrow stromal cells in the epiphysial region of a femur. eb, epiphysial bone. (D) Infection of adrenal medulla. Shown is a pair of huge, plaque-like foci in the medullary parenchyma. (E) Infection of adrenal cortex. Shown is an advanced focus extending from the zona glomerulosa deeply into the zona fasciculata. (F) Few infected glandular epithelial cells were present in the acini of the submandibular gland. d, duct system of salivary gland. (G) Infection of the lungs. Visible are infected cells in the alveolar septa and in the peribronchiolar connective tissue. Other sections show also infected capillary endothelial cells. a, alveolus; b, bronchioli. (H) Infection of cardiac muscle cells. (J) Infection of gastric mucosa. Counterstaining was performed with hematoxylin. Bars represent 25 μm.
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