Rat cytomegalovirus major immediate-early enhancer switching results in altered growth characteristics

Abstract

It has been hypothesized that the major immediate-early (MIE) enhancer of cytomegalovirus (CMV) is important in determining virus tropism and latency because of its essential role in initiating the cascade of early gene expression necessary for virus replication. Although rat CMV (RCMV) and murine CMV (MCMV) exhibit extreme species specificity in vivo, they differ in their ability to replicate in tissue culture. MCMV can replicate in a rat embryo fibroblast (REF) cell line while RCMV does not grow in murine fibroblasts. The tropism is not due to a block in virus entry into the cell. We have constructed a recombinant RCMV in which the RCMV MIE enhancer has been replaced with that of MCMV. Growth of the recombinant virus in tissue culture remains restricted to rat cells, suggesting that other viral and/or host factors are more important in determining in vitro tropism. Unlike findings using recombinant MCMV in which the human CMV (HCMV) MIE enhancer substitutes for the native one (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998), infection with our recombinant virus at a low multiplicity of infection resulted in a substantial decrease in virus replication. This occurred despite comparable or increased MIE transcription from the recombinant virus. In vivo experiments showed that the recombinant virus replicates normally in the spleen during acute infection. Notably, the recombinant virus appears to be deficient in spreading to the salivary gland, suggesting a role for the MIE enhancer in tropism for certain tissues involved in virus dissemination. Four months after infection, recombinant virus with the foreign MIE enhancer was reactivated from spleen explants.

FIG. 1

Generation of a recombinant RCMV (RCMV^MEnh) that contains the MCMV MIE enhancer in place of the RCMV MIE enhancer. The DNA to be inserted includes the MCMV MIE enhancer from bp −31 to −835 relative to the MCMV IE1 cap site (therefore not including the MCMV TATA box) and the lox–β-Gal–lox expression cassette, which allows for identification of recombinant virus. The RCMV^MEnh transfer vector (MEnh TV) contains the MCMV MIE enhancer, a β-Gal expression cassette with lox sequences at each end, flanked by two stretches of RCMV DNA (A and B), which allow for homologous recombination to occur. Region A contains the RCMV DNA sequence from bp −908 to −2404 relative to the cap site of RCMV IE1. Region B contains the RCMV DNA sequence from bp −49 to +1980 relative to the RCMV IE1 cap site and therefore contains the RCMV MIE TATA box-to-cap site region. Successful recombination results in a recombinant RCMV containing the MCMV MIE enhancer and the lox–β-Gal–lox cassette in place of the wt enhancer. Passage through the REF-Cre cell line recombines out the β-Gal expression cassette, leaving one lox site.

FIG. 2

Southern blot analysis of recombinant RCMV. Shown is a Southern blot of wt RCMV (1), RCMV^MEnhβgal (2), RCMV^MEnh (3), and wt MCMV (4) virion DNA digested with HindIII or KpnI and probed with the MCMV enhancer.

FIG. 3

One-step growth curves comparing wt virus, RCMV^MEnh, and RCMV^rep at low (A) and high (B) MOI of REF cells. Virus supernatants were collected postinfection at the times indicated, and titers were determined by standard plaque assay on REF cells. Data presented are averages of three experiments ± standard deviation.

FIG. 4

Real-time quantitative PCR analysis of MIE transcription. Total RNA of cells infected with wt virus, RCMV^MEnh (MEnh), or RCMV^rep (repair) was extracted postinfection as indicated. Following reverse transcription, primers specific for exon 2 and either exon 4 (IE1) or exon 5 (IE2) were used in PCRs with internal exon 4- or exon 5-specific fluorescent probes in a quantitative PCR sequence detector. Results were normalized to those of GAPDH. Cells were infected at a low MOI (A) or at a high MOI in the presence of cycloheximide (B).

FIG. 5

Acute in vivo infections. Because of the variability in organ titers of mice and rats following inoculation with identical viral pools, a mixture of wt virus, RCMV^MEnh, and RCMV^rep with RCMV^βgal was used to assess differences in in vivo replication. RCMV^βgal served as an internal control. Rats were inoculated with the virus pools, and the organs were harvested at the times indicated. Results are expressed as the percent of wt virus, RCMV^MEnh, or RCMV^rep (non-blue plaques) in the mixture, the remainder being RCMV^βgal (blue plaques). Thus, an asterisk at 10% indicates that of the virus isolated from that organ, 10% was wt, RCMV^MEnh, or RCMV^rep and 90% was RCMV^βgal. Not all rats were positive for virus. Spleens were harvested from rats (three per group) 3 days postinfection, and salivary glands were harvested from rats (six per group) 6 days postinfection.

FIG. 6

PCR of spleen DNA from six rats infected with RCMV^MEnh only. Approximately 120 days postinfection, DNA from spleens was used for PCR with primers specific for RCMV^MEnh virus. The Southern blot was probed with an internal oligonucleotide.