And, by inference from the reduction of free MA protein, inAnd, by inference from the

May 17, 2018

And, by inference from the reduction of free MA protein, in
And, by inference from the reduction of free MA protein, in Mo-MLV/Ga-p12. However, the MA/p12 cleavage was also poor for wild type Mo-MLV (Additional file 4, Figure 1D and [8,15,21]). And as completely blocking the cleavage between MA and p12 onlymoderately reduces viral infectivity (less than 10 fold) [16], we think it is unlikely that this accounts for the inability of the N-terminus of GaLV p12 to functionally replace the N-terminus of Mo-MLV p12 (Figure 7A, right panel).p12 does not self associate in vitroMo-MLV p12 protein was purified from E. coli using a GST tag. The protein was soluble at pH 1.8 and when heated at 65 , indicating either highly reversible renaturation/denaturation or that it has little tertiary structure at all, at least when unbound to interaction partners. Multi-angle laser light scattering (MALLS) and analytical ultracentrifugation (AUC) were used to determine that p12 was a single species (Figure 8A) with a narrow distribution of molar mass with no significant concentration dependency (Figure 8A). In order to confirm the MALLS data, quantitative sedimentation equilibrium experiments at multiple rotor speeds and varying sample concentration were undertaken (Figure 8B). No PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26740125 concentration dependency was apparent, so global fitting, incorporating the data from multiple speeds and multiple sample concentrations, was applied to extract a final weight-averaged molar mass of 9.3 ?0.2kD (Figure 8B). The finding that p12 remains a monomer, even at high concentrations, is important information for constructing a model for p12 function, as these data demonstrate thatWight et al. Retrovirology 2012, 9:83 http://www.retrovirology.com/content/9/1/Page 13 ofthe dominant negative phenotype of the C-terminal mutants is unlikely to result from p12 self-association.Figure 8 Biophysical analysis of purified p12 protein. A) SEC-MALLS analysis of p12. Scattered light intensity (solid line) and differential refractive index (dashed line) are order C.I. 75535 plotted against retention time. Traces from p12 samples applied to a G75 (10/30) size exclusion column at 9.0 mg/ml (blue) and 4.5 mg/ml (red) are shown. The molar mass distributions, plotted as points, were determined throughout each peak as described in the text. B) Sedimentation equilibrium analysis of p12. A typical multispeed sedimentation equilibrium profile obtained from interference data collected on p12 at 100 M is shown. The data were collected at 20, 25 and 30 krpm (red, green and blue points respectively). A global fit single-species ideal solution model incorporating data from all speeds and concentrations (black lines) gives a p12 molar mass of 9.3kD.Discussion The early post-entry stages of the retroviral life cycle are targets for several restriction factors and antiretroviral drugs [2,26]. However, many of these steps, such as uncoating, trafficking and nuclear localization are poorly defined. A better understanding of the processes and interactions that occur during early infection may lead to novel therapeutics for retroviral infections and improved retroviral vector design. In 1999, Yuan et al. showed that the p12 protein of MLV was essential during the early stages of MLV replication [8] and a later study identified “footprints” in p12 where insertions inhibited viral infectivity [9]. However, the function of p12 has remained obscure. Further PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 studies showed no gross differences in PIC structure between wild type and mutant virions [10], and the mutant PICs tested were compet.