[PubMed] [Google Scholar] 35. very low capsid protein transcription reduced the potential particle production to insignificant levels. These effects can be ameliorated by adding human cytomegalovirus tegument proteins and immediate-early protein 1. They function synergistically to produce significant amounts of mCMV in human cells. While the possibility that mouse cytomegalovirus might replicate in human cells raises caution in the use of this virus as a transgene vector, manipulation of the mouse cytomegalovirus genome to allow limited spread to other human cells might also provide an advantage for the distribution of certain transgenic products. Cytomegaloviruses (CMV) are a widespread subgroup of should alleviate these blocks and lead to viral particle production. To take into consideration the possibility of low-level transmission of viral genomes short of plaque formation, human astrocytoma cell line U373 and its constitutively IE1-producing derivative, U373-IE1, as well as human fibroblasts and the derivative producing IE1 (ihfie1.3) were infected with GFP-tagged mCMV at low (0.1) PFU (as determined on 3T3 cells). Comparison of cultures infected at confluence showed that 3T3 cells and HF were equally infected (Fig. 4A and B), indicating equal transmission of viral genomes to the nucleus and equal activation of the major immediate-early promoter (MIEP) in these two fibroblast lines. Surprisingly, the human U373 cells had a much higher apparent contamination rate at the same particle input and the number of U373-IE1 cells producing MIEP-driven GFP at 24 h p.i. was even higher than that of the U373 cells (Fig. 4C and D). Assuming the same level of contamination, the presence of hCMV IE1 appears to activate the mCMV MIEP in more cells, consistent with Western blot analysis of the actual viral proteins, which also revealed activation of the mCMV MIEP (Fig. ?(Fig.5A).5A). In U373 cells, IE1 expression declined over time and M141 showed the same pattern as for HF, whereas U373-IE1 cells continued to produce IE1, M112/113, and also M141, similarly to 3T3 cells (compare Fig. ?Fig.5A5A and Fig. ?Fig.1).1). Based on the high contamination rate of U373-IE1 cells, we conclude that fibroblasts of both species can totally suppress a considerable amount of infecting virus even at the MIEP level. By use of Southern analysis, the hCMV IE1-expressing cells also produced substantially more mCMV DNA and, importantly, substantially more full-size viral genomes clearly recognized when the time sequence of mCMV DNA production was compared between U373 and U373-IE1 cells, particularly recognizable at late times after contamination (Fig. ?(Fig.5B).5B). However, the presence of hCMV IE1 did Ac-Gly-BoroPro not prevent mCMV Ac-Gly-BoroPro DNA degradation. Open in a separate window FIG. 4. Plaque formation of GFP-producing mCMV contamination in IE1-expressing human cells. Confluent cell lines (indicated at lower left) were infected with equal amounts of mCMV expressing GFP (RVG102), with titers that induced 1% GFP-producing 3T3 cells. Much higher numbers of U373 astrocytoma cells produce GFP (C), and more than 50% of the hCMV IE1-expressing U373 cells were labeled with GFP (D). (E GDF2 to H) Size increase of a plaque over time in U373-IE1 cells; the different days (e.g., D5, day 5) are indicated at the upper right. Open in a separate window FIG. 5. (A) Expression of immediate-early and early proteins in human U373 and U373-IE1 cells at different times (hours) post-mCMV contamination. The membrane was stripped each time before probing with a different specific antibody occurred. Tubulin Ac-Gly-BoroPro was used as a loading control. The gel was loaded with less cell lysate for the U373-IE1 sample. (B) mCMV DNA production in mCMV-infected human U373 and U373-IE1 cells, as determined by Southern blotting. Cells were harvested at different times (hours) after contamination. m, mock infected. To address whether these mCMV protein-producing human cells generate virus that can reinfect additional cells and considering the possibility that reinfected cells might not be detected as plaque formation because of low particle production, we photographed a small cluster of cells over several days (Fig. 4E to.