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. 2003 Mar;77(5):3217-28.
doi: 10.1128/jvi.77.5.3217-3228.2003.

An essential role of the enhancer for murine cytomegalovirus in vivo growth and pathogenesis

Peter Ghazal  1 , Martin Messerle, Kent Osborn, Ana Angulo
Affiliations

An essential role of the enhancer for murine cytomegalovirus in vivo growth and pathogenesis

Peter Ghazal et al. J Virol. 2003 Mar.

Abstract

The transcription of cytomegalovirus (CMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing an array of binding sites for a variety of cellular transcription factors. Previously, using bacterial artificial chromosome recombinants of the virus genome, it was reported that the enhancer region of murine CMV (MCMV) is dispensable but performs a key function for viral multiplication (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998). In the present study, we defined, through the reconstitution of infectious enhancerless MCMVs, the growth requirement for the enhancer in tissue culture and explored its significance for steering a productive infection in vivo. A comparison of cis and trans complementation systems for infection of enhancerless virus in permissive fibroblasts revealed a multiplicity-dependent growth phenotype that is severely compromised in the rate of infectious-virus multiplication. The in vivo impact of viruses that have an amputated enhancer was investigated in an extremely sensitive model of MCMV infection, the SCID mouse. Histological examination of spleens, livers, lungs, and salivary glands from animals infected with enhancer-deficient MCMV demonstrated an absence of tissue damage associated with CMV infection. The lack of pathogenic lesions correlated with a defect in replication competence. Enhancerless viruses were not detectable in major target organs harvested from SCID mice. The pathogenesis and growth defect reverted upon restoration of the enhancer. Markedly, while SCID mice infected with 5 PFU of parental MCMV died within 50 days postinfection, all mice infected with enhancerless virus survived for the duration of the experiment (1 year) after infection with 5 x 10(5) PFU. Together, these results clarify the importance of the enhancer for MCMV growth in cell culture and underscore the in vivo significance of this region for MCMV virulence and pathogenesis.

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Figures

FIG. 1.
FIG. 1.
Construction of enhancerless MCMV BAC genomes. The HindIII map of the MCMV genome is shown at the top. The HindIII K and L fragments from the parental MCMV BAC genome, C3X, are expanded below to show the major IE gene region. The structures of the ie1, ie2, and ie3 transcripts are indicated below the expanded map. Coding exons are shown solid, and the first noncoding exons of the ie1-ie3 and ie2 transcription units are depicted as open rectangles. The shaded box marks the MCMV enhancer ie1-ie3 promoter. The recombinant MCMV BAC plasmids C3XdE, C3XdE::Luc, and C3XdE::Luc-rev, shown below the C3X genome, were generated by successive rounds of homologous recombination in E. coli starting from the parental BAC plasmid C3X (for C3XdE and C3XdE::Luc) or C3XdE::Luc (for C3XdE::Luc-rev), as indicated in Materials and Methods. The MCMV BAC plasmid C3XdE contains a 1.1-kbp deletion within the HindIII L fragment extending from nucleotide position −48 to −1191 relative to the ie1-ie3 MCMV transcription start site. C3XdE::Luc contains a 770-bp fragment from the luciferase gene replacing nucleotide sequences from −48 to −1191 of the MCMV MIEP enhancer region. C3XdE::Luc-rev is a revertant of C3XdE:: Luc in which the enhancer sequences were reconstituted. The 1.1-kbp deletion in the enhancer region is marked Δ. The crosshatched box represents the luciferase stuffer region. The sizes of the natural and new HindIII K and L fragments are indicated. The illustration is not drawn to scale.
FIG. 2.
FIG. 2.
Structural analysis of the enhancerless MCMV BAC genomes. DNAs of the HindIII-digested BAC plasmids C3X (lane 1), C3XdE (lane 2), C3XdE::Luc (lane 3), and C3XdE::Luc-rev (lane 4) after separation on a 0.7% agarose gel and ethidium bromide staining. The HindIII fragment names (9) are shown at the left. The sizes of the natural HindIII L and M fragments and the new HindIII L fragment for each BAC plasmid are indicated with arrows at the right.
FIG. 3.
FIG. 3.
Growth curve analysis of enhancerless MCMV mutants in tissue culture. NIH 3T3 cells (A and B) or NIH 3T3-Bam25 cells (C) were infected at an MOI of 0.01 (A and C) or 3 (B) PFU/cell with parental MCMV, MCMVdE, MCMVdE::Luc, and MCMVdE::Luc-rev. At the indicated time points after infection, supernatants from the infected cultures were harvested, and titers were determined on monolayers of NIH 3T3-Bam25 cells. The error bars indicate the standard deviation of the viral titers from three separate cultures.
FIG. 4.
FIG. 4.
Excision of the BAC vector sequences from enhancerless MCMV genomes. PCRs were performed using as templates genomic DNAs isolated from viral stocks of MCMV (lanes 1 and 6), MCMVdE (lanes 2 and 7), MCMVdE::Luc (lanes 3 and 8), and MCMVdE::Luc-rev (lanes 4 and 9) or from an early MCMVdE::Luc passage (lanes 5 and 10). Two different primer sets were used to examine the correct excision of the BAC vector sequences within the viral genomes. The first primer set contains a primer that anneals with the BAC sequence and a primer located with the EcoRI g fragment (lanes 1 to 5), and the second primer set contains two primers that bind to viral sequences flanking the excision site (lanes 6 to 10). The amplified products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Shown are the products obtained in the PCR. The positions of the size markers are shown at the left. The sizes of the amplified products are indicated by arrows.
FIG.5.
FIG.5.
Histological examination of livers, spleens, lungs, and salivary glands from mice infected with enhancerless MCMV. Shown are sections from livers (A to D), spleens (E and F), lungs (G and H), and salivary glands (I and J) of CB17 SCID mice 14 days after infection with 6 × 105 PFU of tissue culture-passaged MCMVdE::Luc (A, C, E, G, and I) or MCMVdE::Luc-rev (B, D, F, H, and J). (A, C, E, G, and I) No lesions present. (B) Multifocal inflammatory foci (marked by arrowheads). (D) Note cytomegalic cell with inclusion (arrow). The number of inflammatory foci present in liver sections from MCMVdE::Luc-rev-infected animals was 23 ± 10 per mm2. (A and B) Overview; (C and D) details of portions of A and B, respectively. (F, H, and J) Note the cytomegalic cells (indicated by arrows). (F) Neutrophilic infiltrate in splenic white pulp. (H) Lung alveolar septal thickening with fibrin and inflammatory cells. (J) Salivary gland focal necrosis with inflammation. Bars, 400 (for A and B) and 100 (for C through J) μm. Magnification, ×25 (A and B) and ×100 (C through J).
FIG. 6.
FIG. 6.
Growth of enhancerless MCMV mutants in the spleens, livers, and lungs of SCID mice. Groups of SCID mice (CB17; four to six per group) were inoculated intraperitoneally with 6 × 105 PFU of tissue culture-propagated MCMV, MCMVdE, MCMVdE::Luc, and MCMVdE::Luc-rev. The mice were sacrificed on day 7 or 14 postinfection (p. i.), and their spleens, livers, and lungs were harvested for determination of viral titers. The organs were weighed and sonicated as a 10% (wt/vol) tissue homogenate in DMEM. The viral titers of the resulting homogenates were determined on NIH 3T3-Bam25 cells. Titers corresponding to the spleen, liver, and lungs from each individual mouse within a group are shown. The dashed lines represent the limits of detection. The horizontal bars indicate the mean values.
FIG. 7.
FIG. 7.
Attenuation of enhancerless MCMV mutants in SCID mice. Groups of SCID mice (CB17; three to four mice per group) were inoculated intraperitoneally with the indicated amounts of either parental MCMV, MCMVdE, MCMVdE::Luc, or MCMVdE::Luc-rev and monitored daily for survival. One hundred percent of the mice that received 5 × 100, 5 × 101, 5 × 102, 5 × 103, or 5 × 104 PFU of MCMVdE or MCMVdE::Luc survived the infection for the time the experiment lasted (150 days; data not shown).
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