Role of SV40 ST antigen in the persistent infection of mesothelial cells

Abstract

Viral DNA is maintained episomally in SV40 infected mesothelial cells and virus is produced at low but steady rates. High copy numbers of the viral DNA are maintained in a WT infection where both early antigens are expressed. In the absence of ST, cells are immortal but non-transformed and the infected cells maintain only a few copies of episomal viral DNA. We show that ST expression is necessary for the maintenance of high copy numbers of viral DNA and that the PP2A binding ability of ST plays a role in genome maintenance. Interestingly, an siRNA to the virus late region downregulates virus copy number and virus production but does not prevent the anchorage-independent growth of these cells. Furthermore, addition of virus neutralizing antibody to culture media also decreases copy numbers of viral DNA in WT-infected cells, suggesting that virus production and re-infection of cells may play a role in maintaining the persistent infection.

Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis

“…The circular SV40 genome has been shown to be maintained in certain intracranial tumors, but Krieg et al [161] suggest that SV40 is not the cause of these human tumors. It remains unknown which protein forms the physical connection between the SV40 episome and host chromosomes [162].…”

SV40 reporter viruses

“…In our studies, this virus has been useful for detecting superinfection of persistently infected mesothelial cells, a strategy that was used most recently in evaluating the role of the ST antigen in maintaining high copy numbers of the SV40 genome (Fahrbach et al, 2008). The construction of reporter viruses was undertaken initially because different clones and sublines of persistently infected human mesothelial cells appeared to respond differently to a superinfecting virus.…”

“…These cells express LT and ST from integrated viral DNA that cannot replicate because of a defective origin of replication. Some experiments were done using 5ADL, a persistently infected human mesothelial cell culture (Fahrbach et al, 2008;Yu et al, 2001) that contains episomal DNA of the virus DL-888 (Shenk et al, 1976). DL-888 expresses LT but not ST because of a small deletion across the ST splice donor sequence.…”

“…The tagged viruses made in this study were of particular use for monitoring superinfection of persistently infected cells lines in which all cells already express LT. To show that superinfecting virus can be differentiated from virus already present in SV40 transformed mesothelial cells, serial dilutions of vC-LT FLAG SV40 were used to infect 5ADL cells, an immortalized human mesothelial cell line that expresses LT in every cell because of persistent infection with DL-888 [Fahrbach, 2008. At 36 hours post-infection, cells were assayed for the expression of the FLAG tag (vC-LT FLAG -infected), as well as for the resident DL-888 virus using PAB419 to detect LT (Crawford et al, 1982;Harlow et al, 1981).…”

“…Persistently infected cultures contain far fewer genomes of SV40 per cell (50-1000 molecules per cell), 1-2 orders of magnitude lower than the number observed during permissive infection. ST expression is required to maintain this high copy number, and cells that lack ST contain only very low levels of episomal viral DNA (Fahrbach et al, 2008). Persistently infected cultures produce low but detectable levels of infectious virus continually in the absence of cytopathic effect (CPE).…”