A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques

Abstract

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.

FIG. 1.

(A) Configuration of the plasmids used in this study. The pUC19 sequences in each plasmid are shown as a thin line, and the viral sequences are presented as a thick line. For the construction of pWC133, a loxP adapter was inserted into the BglII site of pWC131. (B) Schematic diagram of viral genome structure with expansion of the region from US1 to US3 of RhCMV and RhCMV-EGFP. The locations of the EGFP expression cassette flanked by two loxP sites and the additional NotI/SalI sites in the viral genome introduced by homologous recombination are shown. The square represents the junction region between UL and US. Arrows indicate the locations of diagnostic PCR primers PAB431 and PAB435 specific to US1 and US2, respectively.

FIG. 2.

Analyses of RhCMV and RhCMV-EGFP genome organizations. (A) Diagnostic PCR for RhCMV and RhCMV-EGFP isolates using supernatants collected from virus-infected cultures as templates. (B) Gel electrophoresis of SalI- and NotI-digested viral nucleocapsid DNA. Novel restriction fragments generated following recombination are marked with asterisks. The loss of the NotI restriction fragment is marked with an arrow. Size standards are displayed on the left of the gel pictures and are indicated in kilobases. Lane M, DNA marker; lane U, uninfected control; lane 1, wild-type RhCMV; lane 2, plaque-purified RhCMV-EGFP; lane 3, virus recovered from fetus 3; lane 4, virus recovered from fetus 4.

FIG. 3.

Replication kinetics of RhCMV-EGFP and EGFP expression in infected cells. (A) The multiple-step growth curve of RhCMV-EGFP is compared to that of its parental RhCMV strain. Telo-RF cells were infected in triplicate with each virus at an MOI of 0.01. Supernatants and cells were collected longitudinally from the infected cultures for standard plaque assays and FACS analyses, respectively. The titers of infectious virions in the samples are shown as a solid line. The percentages of GFP-positive cells in total gated populations are presented as bars with values shown. Data points represent the mean of three independent cultures, with the standard deviations indicated by error bars. (B) Overlaid histogram of fluorescence cytometry profiles of cells infected with either RhCMV (thick line) or RhCMV-EGFP (thin line). Mock-infected cells are also shown (shaded). Telo-RF cells were infected at an MOI of 0.01, collected on 4 dpi, fixed with 1% paraformaldehyde, and analyzed by FACS.

FIG. 4.

3′ RACE analyses of RhCMV-EGFP gene expression profiles. (A) US1 and US3 expression levels in RhCMV- or RhCMV-EGFP-infected Telo-RF at different time points (indicated in hours) after virus inoculation. (B) Constitutive expression of EGFP open reading frame and the temporal regulation of viral IE2 and US2 genes in RhCMV-EGFP-infected cells. Cytoplasmic RNA was isolated from infected cultures at different time points in the presence (+) or absence (−) of either 200 μg of cycloheximide/ml (12 hpi) or 400 μg of phosphonoformic acid/ml (24 and 48 hpi). 3′ RACE for GAPDH was performed as an internal control. Size standards are displayed on the left of the gel pictures and are indicated in base pairs. Lane M, DNA marker; lane U, uninfected control.

FIG. 5.

PCR-RFLP analyses of RhCMV and RhCMV-EGFP of different passages. PCR amplicons derived from primers within US1 and US2 were digested with four- or five-base cutters. Digested fragments were separated by electrophoresis on 2% Metaphor agarose gels and visualized by ethidium bromide staining. Size standards are displayed on the left of the gel pictures and are indicated in base pairs. Lane M, DNA marker; lane W, wild-type RhCMV; lane 1, RhCMV-EGFP passage 6; lane 2, RhCMV-EGFP passage 18.

FIG. 6.

Distribution of RhCMV-EGFP-infected cells in fetal brain. Tissues were collected from fetus 2 (F to I) and fetus 4 (A to E) at 10 and 23 dpi, respectively. Immunostaining was carried out using the polyclonal antibody for GFP (A and B) or RhCMV IE1 (C to I). (A and C) Serial brain sections with periventricular foci stained with different antibodies; (B) cells in the meninges are labeled after immunoperoxidase staining of GFP; (D) cerebral neocortex; (E) cerebellum; (F) tectal neuroepithelium; (G) choroid plexus; (H) striatal neuroepithelium; (I) cerebral neocortex. CP, cortical plate; GL, germinal layer; IZ, intermediate zone; ML, marginal layer; MZ, marginal zone; SZ, subventricular zone; VZ, ventricular zone. Bars, 50 μm.

FIG. 7.

Immunoperoxidase staining of tissues collected from fetus 4, using polyclonal antibody for RhCMV IE1 (A to D) or GFP (E). (A) Infected cells adjacent to the endothelial cells around the portal vein were frequently found in the liver. (B) Infected cells in developing glomeruli and mesenchyme of the kidney. (C) In the duodenum, stained cells were observed in the lamina propria. (D and E) Mesenchymal cells in the lung were highly infected. Bars, 50 μm.

FIG. 8.

Fluorometric detection of EGFP expression under the control of SV40 early promoter from brain sections from fetus 3 (B and D) and nuclei of cells counterstained with DAPI (A and C). Strong RhCMV-infected regions were found in the periventricular area. Some infected cells scattered in the cerebral cortex were also noted. Arrows indicate individual infected cells that had weaker DAPI staining but enlarged nuclei. GFP-positive cells were frequently surrounded by green halos, presumably from diffusing EGFP of adjacent cells caused by cell lysis or sectioning. Bars, 200 μm (A and B) and 25 μm (C and D).