An Integrated Approach to Elucidate the Intra-Viral and Viral-Cellular Protein Interaction Networks of a Gamma-Herpesvirus
Figure 5
Functional validation of cellular proteins from the MHV-68-cellular protein interaction network.
A) Overview of the functional validation approach used to evaluate the effect of cellular interacting proteins on MHV-68 replication. HEK293T cells were reverse transfected with siRNAs targeting cellular genes and infected with M3-Luc MHV-68, which provides a sensitive and conveniently assayed measure of virus replication. The supernatant was collected at 50 and 58 h post-infection and used to infect fresh 293T cells. Luciferase activity was measured at 20 h post-infection. (B) The effect of inhibiting expression of cellular proteins from the Y2H-High priority group (Y2H-HP) on MHV-68 replication. Expression of the 60 highest scoring cellular proteins was inhibited using a single siRNA per gene and MHV-68 replication was assessed as described above. The percentage of siRNAs that enhanced, inhibited, or had no effect on MHV-68 replication is shown. (C) Cellular proteins with high priority scores were more likely to affect MHV-68 replication. Twenty proteins were randomly selected from the Y2H-HP group, the Y2H-low priority and not scored groups (Y2H-LP/NS), and the group of cellular proteins not known to interact with MHV-68 proteins (C-R). The expression of each protein was inhibited with two siRNAs that targeted different regions of the cognate mRNA. MHV-68 replication in the siRNA-transfected cells was measured as described above. Graph shows the percentage of proteins from each group that caused a statistically significant enhancement or inhibition of MHV-68 replication. Asterisks indicate the difference between groups was statistically significant (p<0.05). (D) GO term analysis of high scoring cellular proteins identified in the Y2H screen.