Figure 4

Down-regulation of SMC marker genes in response to XMRV-infected LNCaP conditioned media is dependent on the presence of XMRV envelope proteins. A. The env genes of 4070a and XMRV were swapped to form chimera retroviruses that were used to infect LNCaP cells in equal viral titers. XMRV-4070a-env was cloned by replacing the env gene of XMRV VP62 with the env gene of MoMLV-4070a. 4070a-XMRV-env was cloned by replacing the env gene of MoMLV-4070a with the env gene of XMRV VP62. Both chimera viruses (XMRV-4070a-env and 4070a-XMRV-env) represent fully infectious, replication competent viruses (Additional file 4: Figure S4). TCM was generated from the resulting tumor cells including uninfected LNCaPs, and MoMLV-4070a-, XMRV-, XMRV-4070a-env-, or 4070a-XMRV-env-infected LNCaP cells. Rat aortic SMCs were cultured in TCM for 72 hours followed by q-rtPCR analysis of expression of SMC differentiation marker genes including Tagln and Myh11. B. Q-rtPCR for expression of smooth muscle marker genes Tagln and Myh11 in rat aortic SMCs after culture in TCM of uninfected LNCaPs, LNCaps infected with replication competent viruses MoMLV-4070a or XMRV, or MoMLV pseudotyped with XMRV envelope, amphotropic envelope or PreXMRV-1 envelope and containing only the GFP gene, demonstrating that SMC marker gene down-regulation when cultured in the TCM of LNCaPs infected with XMRV is dependent on the presence of the XMRV env proteins. C. Q-rtPCR for expression of SMC marker genes Tagln and Myh11 in rat aortic SMCs after culture in the TCM of LNCaPs that were infected with XMRV or MoMLV-4070a viral particles that were exposed to 0, 10, 20 or 40 minutes of UV irradiation, demonstrating that LNCaP cells that are cultured with XMRV particles that are exposed to UV irradiation at doses that inactivate viral infectivity (Additional file 5: Figure S5) produce TCM that suppresses marker gene expression in rat aortic SMCs.