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.

Figure 1. Detection of viral DNA in mesothelial cell lines

Total genomic DNA was isolated from 5ADL and 5AWT cells as described in Materials and Methods. DNA from approximately 5×10⁵ cells was linearized with EcoRI and run on an agarose gel for analysis by Southern blotting. Viral DNA was detected by incubation with a probe made from HindIII-digested SV40 DNA. (A) The membrane was exposed until signal was detected in 5AWT samples. Control lanes are loaded with 25, 2.5 or 0.25ng of linear SV40 plasmid. This represents a range between 5×10⁹ and 5×10⁷ total copies of SV40. Inset: A ten-times longer exposure of the 5ADL lanes shown in A. (B) Genomic DNA was prepared from 5AWT cells or a clone of 5ADL, clone 4B5. DNA was digested with XbaI (X) or BglII (B), enzymes that do not cut SV40, or with EcoRI (E), which cuts SV40 once. The positions of form I and nicked circular SV40 DNA are makes as uncut. The position of linear SV40 DNA is also shown. After digestion, 5AWT DNA was diluted 20-fold before running on the same agarose gel as the DNA from the 5ADL clone.

Figure 2. Maintenance of the SV40 genome in super-infected mesothelial cells

5ADL cells were infected at 10 pfu/cell with either wild-type SV40 (WT) or dl888, a mutant form of the virus that does not express small-t antigen. Cells were infected for two days and then passaged at a 1:10 dilution once per week. Hirt extracts were collected on passages 1, 3, 5, and 7. Equal amounts of DNA from each sample were linearized with EcoRI and analyzed by Southern blotting. Viral DNA was detected by incubation of the membrane with a probe directed toward SV40 DNA. (A) Low molecular weight DNA was analyzed after 2 days of infection or following the third passage. Three times as much DNA was loaded in the second lane of each sample. (B) A clone (ST#4) of 5ADL that stably expressed small-t antigen was generated as described in Materials and Methods. This clone and 5ADL parental cells were infected with dl888 virus, then passaged weekly. Amounts of low molecular weight DNA from passage 1, 3, 5 and 7 were compared.

Figure 3. ST is required for maintenance of a superinfecting DL888 genome

5A97 cells were prepared by infecting mesothelial cells with the ST mutant C97S. Although this mutant is less defective functionally than C103S, it was used because the mutant ST is quite stable. (A) ST protein expressed by 5A97 was compared to that of 5AWT by western blotting using monoclonal antibody Pab419. (B) 5A97 cells were superinfected with either dL888 or WT SV40 at 10 pfu/cell, then passaged at weekly intervals as described in Figure 2. Real-time PCR analysis was performed on Hirt extracts prepared at passage 11.

Figure 4. Depletion of genome copy number does not prevent anchorage-independent growth of the mesothelial cells infected with WT virus

5AWT cells were infected with lentivirus expressing shRNA against the late region transcript of SV40 and the puromycin resistance gene. Following puromycin selection, surviving cells (5AWT/si) were pooled and analyzed. (A) The viral load produced by 6 cm dishes of 5AWT and 5AWT/si cells was determined by a plaque assay. CV1 cells were infected with undiluted and diluted (1:10 and 1:100) virus preparations made from equal numbers of cells. The calculated titer is shown below each column. (B) Cells were harvested and lysates were immunoblotted for the presence of VP1, LT, and GAPDH. (C) To detect ST, extracts of ³⁵S-methionine labeled cells were immunoprecipitated with pAB419, then immunoprecipitated protein was detected following SDS-PAGE by autoradiography. (D) Anchorage independent growth was determined by plating 2.5 × 10⁵ 5ADL, 5AWT, or 5AWT/si cells in 1.8% methylcellulose in agarose coated Petri dishes.

Figure 5. Growth in SV40 neutralizing antibody decreases viral copy number in WT infected mesothelial cells

A. 5AWT cells were grown in the presence of media containing 1% neutralizing antibody or media alone. The cells were passaged and collected for real-time PCR once a week for two weeks. B. After 2 weeks in culture, one set of cells previously grown with the neutralizing antibody was washed and allowed to grow in media without antibody for four more weeks. Real-time PCR was performed weekly, but only the levels of DNA found after the full four-week recovery period are shown. The solid bar represents cells that were grown without antibody for the entire six-week period. Real-time PCR was performed in triplicate and standard error is indicated. Copies of SV40 per cell were calculated by comparison to known concentrations of SV40 DNA.