Supplementary MaterialsAdditional file 1 Activity of p12 mutants in a panel

Supplementary MaterialsAdditional file 1 Activity of p12 mutants in a panel of cell lines. synthesized in 293T cells and used in TRIM5alpha and Fv1b abrogation assays (Physique ?(Physique4A4A and ?and4B).4B). Equivalent RT-units of VLPs were used to SB 525334 enzyme inhibitor challenge D17, TE671 and B3T3 cells and infectivity was measured by detection of Cgalactosidase activity in a chemiluminescent reporter assay. Infectivity is usually plotted as a percentage of the B-MLV control. (B) The infectivity of N/Mo LacZ tester viruses that were utilized for TRIM5alpha abrogation assays (Physique ?(Physique4C)4C) was tested in D17 and TE671 cells as in (A). Infectivity is usually plotted as the percentage of the Mo-MLV control. (C) The infectivities of the mixed particles N6/B, B6/N and N6/N and the control viruses that were utilized for TRIM5alpha abrogation assays (Physique ?(Figure4D)4D) were tested in D17 and TE671 cells as in (A). 1742-4690-9-83-S2.tiff (716K) GUID:?C5E13C58-07DA-4F8A-9B55-775D69CE452B Additional file 3 Quantification of viral cDNA levels in D17 cells. Wild type and mutant SB 525334 enzyme inhibitor N-MLV VLPs were produced in 293T cells by transient transfection and equivalent RT-units of VLPs were used to challenge D17 cells. Total DNA was isolated at numerous times post contamination as indicated, and the relative amounts of second strand extension were measured using qPCR. Results are representative of three impartial experiments. 1742-4690-9-83-S3.tiff (144K) GUID:?031A01B4-87B3-41BB-AB74-FA0882A65F15 Additional file 4 Immunoblot analysis of Mo-MLV/GaLV chimeras. LacZ-encoding Mo-MLV/GaLV p12 chimeric VLPs (A) and GaLV/Mo-MLV p12 chimeric VLPs (B) were produced by transfection of 293T cells (as in Figure ?Physique7).7). Equivalent RT-units of particles were concentrated through 20% (w/v) sucrose cushions and lysed in SDS loading dye. Viral proteins were separated on a 10% polyacrylamide gel by SDS-PAGE and p12 was detected with an anti-MLV p12 polyclonal antibody (A and B, top panels) and an anti-MLV p12 monoclonal antibody (A, middle panel; B, bottom panel). The approximate sizes of SB 525334 enzyme inhibitor p12 and MA-p12 are indicated. GaLV p12 is not detected with either of these antibodies. In addition, the Mo-MLV based chimeras were probed with anti-MLV CA and anti-MLV MA antibodies (A, bottom panels). These antibodies did not cross react with GaLV Gag proteins. 1742-4690-9-83-S4.tiff (1.1M) GUID:?0D34B281-E739-48D5-9B0C-D9AB8FBFE6DA Additional file 5 Site directed mutagenesis primer sequences used to construct different gammaretroviral p12 alanine substitution mutations. 1742-4690-9-83-S5.xlsx (44K) GUID:?8C4AF6E4-DE66-453A-A185-B5EBAEF03FB8 Additional file 6 Site directed mutagenesis primer sequences used to construct single amino acid changes in p12. 1742-4690-9-83-S6.xlsx (35K) GUID:?7CECB602-EF50-4749-A0ED-84A72760791C Abstract Background The Moloney murine leukaemia virus (Mo-MLV) gene encodes three main structural proteins, matrix, capsid and nucleocapsid and a protein called p12. In addition to its role during the late stages of contamination, p12 has an essential, but undefined, function during early post-entry events. As these stages of retroviral contamination remain poorly comprehended, we set out to investigate the function of p12. Results Examination of the infectivity of Mo-MLV virus-like particles containing a mixture of wild type and mutant p12 revealed that this N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function, and that the N-terminal activity precedes the C-terminal activity in the viral life cycle. By creating a panel of p12 mutants in other gammaretroviruses, we showed that these domains are conserved in this retroviral genus. We also undertook a detailed mutational analysis of each domain name, identifying residues essential for function. These data show that different regions of the N-terminal domain name are necessary for infectivity in different gammaretroviruses, in stark SB 525334 enzyme inhibitor contrast EIF4EBP1 to the C-terminal domain name where SB 525334 enzyme inhibitor the same region is essential for all those viruses. Moreover, chimeras between the p12 proteins of Mo-MLV and gibbon ape leukaemia computer virus revealed that this C-terminal domains are interchangeable whereas the N-terminal domains are not. Finally, we recognized potential functions for each domain name. We observed that particles with defects in the N-terminus of p12 were unable to abrogate restriction factors, implying that their cores were impaired. We further showed that defects in the C-terminal domain name of p12 could be overcome by introducing a chromatin binding motif into the protein. Conclusions Based on these data, we propose a model for p12 function where the N-terminus of p12 interacts with, and stabilizes, the viral core, allowing the C-terminus of p12 to tether the preintegration complex to host chromatin during mitosis, facilitating integration. site was launched into corresponding to.