Human cytomagalovirus IE1 and IE2 proteins are mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the adenovirus E1A proteins

Abstract

Some epidemiological studies have suggested a possible link between human cytomegalovirus (HCMV) infection and various malignancies, and HCMV has been shown to transform cultured cells. However, viral DNA is not detected in most transformants, and the mechanism by which HCMV might contribute to oncogenesis has remained obscure. Here we show that the HCMV immediate early 1 and 2 genes can cooperate with the adenovirus E1A gene to generate transformed foci of primary baby rat kidney cells. HCMV gene expression is transient and viral DNA is not present in clonal cell lines derived from the transformed foci. We find that the HCMV immediate early proteins are mutagenic, and we propose that HCMV has the potential to contribute to oncogenesis through a "hit-and-run" mechanism, by inducing mutations in cellular genes.

Figure 1

The HCMV IE1 and IE2 genes cooperate with the adenovirus E1A gene to transform primary BRK cells. (A) Representative BRK transformation assay showing the number of foci that arise in response to transfection with different combinations of expression plasmids. (B) BRK transformation assays demonstrating that IE1 plus IE2 cooperate with E1A for transformation. The number of foci reported is the total from 10 plates assayed in four independent experiments. (C) Representative experiment showing that IE1 and IE2 cDNAs cloned in an antisense orientation (αIE1 and αIE2) have no transforming activity. The number of foci reported is the total from three 100-mm plates. In all experiments, the gene products expressed by transfected plasmids are listed below the bars. Cultures transfected with pCGN containing no insert are designated vector.

Figure 2

HCMV IE1 and IE2 genes and their products are not present in transformed BRK cell lines. (A) Lack of expression of the IE1 or IE2 protein in transformed BRK cell lines when assayed by immunoprecipitation of ³⁵S-labeled cell extracts using monoclonal antibody mAB810. A-1 is a cell line established by transfection with the E1A-expressing plasmid alone. A/1/2–1 to A/1/2–10 are cell lines transformed by E1A plus IE1 and IE2. Lanes designated HCMV INF display immunoprecipitates from HCMV-infected human foreskin fibroblasts; lane 13 is an exposure 1/8 as long as that presented for lanes 1–12. Numbers to the left of the gel mark the positions of marker proteins whose sizes are indicated in kDa. (B) Southern blot analysis showing the lack of IE1- or IE2-specific DNA in E1A/IE1/IE2-induced transformants (lanes 1–10). A/B-1 and A/B-2 are cell lines transformed with E1A and E1B-19-kDa. HeLa, 293, and HP1.14 (a derivative HeLa cells expressing IE1) are included as controls. pCGN-IE1 and pCGN-IE2 are IE1 and IE2 expression plasmids, respectively, in amounts equivalent to the rest of the samples, assuming they contain one copy of each of the HCMV genes per cell. Numbers to the left of the gel are marker DNA sizes in kb. (C) Expression of E1A protein as determined by immunoprecipitation followed by immunoblot analysis, both with monoclonal antibody M73 to E1A. Lanes are labeled as in A and B. Numbers to the left of the gel are marker protein sizes in kDa. Autoradiograms were scanned and cropped using Photoshop and figures were prepared using Freehand software.

Figure 3

The IE1 and IE2 gene products are expressed transiently during the transformation of BRK cells. BRK cells seeded on coverslips were transfected with expression plasmids for E1A and E1B-19-kDa (Left) or plasmids for E1A and IE1 and IE2 (Right). At 2, 5, 13, and 19 days after transfection, two coverslips from each transfection were fixed and kept in phosphate-buffered saline until coverslips for all time points were collected. Expression of the transfected genes was examined by indirect immunofluorescent staining with appropriate monoclonal antibodies. Anti-E1A, anti-E1B-19-kDa, and anti-IE1/2 identify the antibodies used for the immunofluorescent staining. Images are from independent coverslips. The images from days 13 and 19 display transformed foci whose cells were positive for E1A and E1B-19-kDa proteins, but completely negative for IE1 and IE2 protein expression. (×50).

Figure 4

The IE1 and IE2 gene products increase the frequency of mutation within the aprt (A) and hprt (B) genes. D422 cells were transfected with expression plasmids (identified below bars). After an incubation period of 4 days, aprt−/− and hprt−/− mutants were selected by the addition of 200 μM diaminopurine or 20 μM 6-thioguanine, respectively, to otherwise purine-free medium. The frequencies of mutation at the two loci for cells exposed to 0.2 mg/ml EMS for 18 hr are also shown.

Figure 5

BRK cells transformed by E1A, IE1, and IE2 often contain mutant p53 alleles. (A) Immunoprecipitation of ³⁵S-labeled cell extracts with a mutant p53-specific antibody, pAb240. 10(1)val5@39°C indicates 10(1) cells harboring a temperature-sensitive form of p53 cultured at 39°C. The left-most lane displays marker proteins whose sizes in kDa are indicated to the left of the autoradiogram. (B) Identification of missense mutations in three E1A/IE1/IE2 transformants. Two independent PCR amplifications of the central DNA-binding domain of p53 were performed for each cell line, and two clones were isolated and sequenced from each amplification reaction. The A/1/2–3 and A/1/2–10 cell lines contain both mutant and wild-type p53 sequences, indicating that only one allele is mutated within the DNA-binding domain. All clones from A/1/2–8 contained a mutation, indicating that both alleles might be altered in these cells.