Characterization of the high Level expression phenotype of the cell Line PCX5 and Studies on the Regulation of rotaviralgene expression in mammalian cells

Abstract

Part I - PCX5 Cell Line and High-Level Gene Expression PCX5, a cell line accidentally generated from the prostate cancer cell line PC3BM, showed high-level expression of PDGF-B and reporter genes ( -gal and GFP) compared to its parental line.

Mechanism of Expression:

Analysis of mRNA and protein levels in PCX5, PC3BM, HeLa, and 293T suggested that the high-level expression phenotype is due to translational upregulation, not transcriptional differences.

Microarray analysis identified 94 differentially expressed genes (59 upregulated, 35 downregulated), but silencing these genes did not significantly affect reporter protein expression.

mTOR Pathway Analysis:

mTOR target proteins 4EBP1 and S6 were phosphorylated at higher levels in PCX5 compared to PC3BM and HeLa.

eEF2K phosphorylation was also elevated, while p70S6K phosphorylation showed no significant difference.

Among pathways activating mTOR (Akt, ERK MAPK, PKC-PA), PKC-PA-mediated activation was predominant in PCX5.

Comparison with 293T revealed that while both use PKC-PA, 293T also activates Akt and ERK MAPK.

These findings highlight the critical role of PKC-PA-mTOR signaling in the high-expression phenotype of PCX5.

Part II - Regulation of Rotaviral Gene Expression Rotavirus, the leading cause of viral gastroenteritis, is responsible for ~600,000 deaths annually, with India accounting for ~25%.

Gene Expression in Transfected Cells:

Rotaviral mRNAs were transcribed at comparable levels in PCX5, HeLa, MA104, and 293T.

Viral proteins NSP5, VP6, and NSP4 were detectable only in PCX5 and 293T, though at low levels.

NSP2 and NSP3 were not detectable in any cell line.

These results suggest negative translational regulation of rotaviral gene expression in mammalian cells, which is deregulated during infection.

Signaling Pathways During Infection:

Insoluble fractions (viroplasms) contained cellular proteins MEK1/2 and Hsp70.

Rotavirus infection activated both Raf/MEK/ERK MAPK and PI3K/Akt pathways, enhancing translational efficiency via mTOR.

Inhibition of MEK1/2 reduced viral protein synthesis, confirming the essential role of the ERK MAPK pathway.

mTOR activation was evident from phosphorylation of 4EBP1, S6, p70S6K, and eEF2K.

Hsp70 Interactions:

Hsp70 was recruited into viroplasms and shown to interact with viral structural proteins VP1 (RdRp) and VP4 (outer capsid spike protein).

The functional role of these interactions in viral replication remains to be elucidated.