Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus

White, Timothy M.; Stanfield, Brent; Bonavita, Cassandra M.; Rudd, Jared S.; Cardin, Rhonda D.

doi:10.1371/journal.pone.0265479

Cited by 1 publication

(7 citation statements)

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“…A second mutant virus, ΔM33-US28+ (hereafter referred to as US28+) in which the M33 ORF is replaced with the HCMV US28 gene, was also used. Wild-type KP, ΔM33 stop, and US28 + MCMV were grown in mSGM salivary gland mesenchymal-like cells ( 35 ) ( Figure 1A ) and NIH 3T3 fibroblasts ( Figure 1B ). All three viruses replicated to similar levels and no significant difference in growth was detected in either mutant as compared with KP (p>0.1).…”

Section: Resultsmentioning

confidence: 99%

“…In order to confirm that the IE1- and m164- specific tetramer staining did in fact correspond to matching T cell functionality, leukocytes were isolated from the spleens of mice acutely or latently infected with KP, ΔM33 stop , or US28 + MCMV. To measure ex-vivo cytotoxicity, splenic leukocytes or magnetic activated cell sorting (MACS)-enriched T cells were added to cultures of a salivary gland-derived mesenchymal-like cell line, mSGM, which we previously isolated and described ( 35 ). These target cells were selected due to their expression of all 3 isotypes of MHC class I which are found in BALB/c mice.…”

Section: Resultsmentioning

confidence: 99%

“…Cytotoxicity was measured in real-time using a non-toxic fluorescent caspase-3/7 substrate, NucView488 (a fluorogenic dye coupled to the caspase DEVD cleavage recognition site) on an Incucyte live-cell imaging system. To selectively present the m164 peptide epitope, a stable lentivirus-transduced cell line, mSGM-m164, was used as previously described ( 35 ). Although we attempted to create a stable cell line expressing IE1 using the same lentivirus backbone, we found that IE1-transduced cells failed to proliferate and died off after a few days.…”

Section: Resultsmentioning

confidence: 99%

“…Although we attempted to create a stable cell line expressing IE1 using the same lentivirus backbone, we found that IE1-transduced cells failed to proliferate and died off after a few days. To remedy this, we used an adenovirus carrying the IE1 open reading frame, Ad5-MCMV_IE1, to induce transient IE1 expression ( 35 ).…”

Section: Resultsmentioning

confidence: 99%

“…Cells were incubated in 5% CO 2 at 37°C. SV40-immortalized mouse salivary gland mesenchymal cells [mSGM cells ( 35 )] were maintained in complete medium as above with the following modifications: FBS concentration was lowered to 5% and an additional 50µM G418 (Gibco) was added to maintain selection.…”

Section: Methodsmentioning

confidence: 99%

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The CMV-encoded G protein-coupled receptors M33 and US28 play pleiotropic roles in immune evasion and alter host T cell responses

et al. 2022

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IntroductionHuman cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation.MethodsMice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop.ResultsWe found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion.DiscussionOur results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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