Figure 1.
Overview of the generation and validation of the MHV-68 intra-viral and virus-cellular protein-protein interaction networks.
Figure 2.
The network of interactions between MHV-68 proteins.
(A) The MHV-68 intra-viral protein interaction network. Rectangles designate viral proteins essential for MHV-68 lytic replication; circles, nonessential MHV-68 proteins. Colors indicate protein functions (blue, DNA replication complex; red, regulatory proteins; orange, envelope proteins; purple, capsid; magenta, assembly or egress; white, unknown function). Arrows point from baits to preys. Interactions confirmed by co-immunoprecipitation (co-IP) are shown as solid lines, whereas those found only by Y2H are shown as dashed lines. Green lines indicate novel interactions found in this study; blue lines represent interactions detected between homologues in other data sets (see Table S1 for references). (B) ORF33 and ORF45 co-localize in the nucleus. Expression plasmids encoding FLAG epitope-tagged ORF33 and YFP-tagged ORF45 were co-transfected into NIH 3T3 cells. FLAG-ORF33 was detected by IFA with anti-FLAG-M2 primary antibody and Alexa Fluor 594-conjugated anti-mouse IgG antibodies. Panel 1 shows FLAG epitope-tagged ORF33 (red); panel 2, YFP-ORF45 (green); panel 3, merged image; panel 4, phase contrast image of same field; panel 5: DAPI-stained nuclei. (C) Co-IP of ORF33 and ORF45. FLAG epitope-tagged ORF33 and V5 epitope-tagged ORF45 were co-expressed in 293T cells, immunoprecipitated with either control IgG, anti-FLAG M2 or anti-V5 antibodies, and subjected to western blotting with anti-V5 antibody (upper panel) and anti-FLAG (bottom panel). I indicate input.
Figure 3.
The MHV-68-cellular protein interactome.
Viral proteins (rectangles) are displayed as a ring and are grouped according to their functional annotation (colors are as described in legend to Fig. 2). Cellular proteins (circles) that interacted with one viral protein are located outside of the ring, whereas those that interacted with multiple viral proteins are located inside. Cellular proteins are colored according to the priority rank, with unranked proteins are shown in white. The size of the circle indicates the magnitude of the effect on MHV-68 lytic replication caused by inhibiting the expression of the cellular gene by RNAi. Large circles imply greater effect; small circle, no effect or not tested. Font color of the cellular protein label indicates the direction of the effect on MHV-68 lytic replication (red, inhibiting expression of the cellular gene increased replication, dark green; inhibiting expression of the cellular gene decreased replication, black, no effect or not tested).
Figure 4.
Network context of cellular proteins that interacted with MHV-68 proteins.
(A) Distribution of the network distances between cellular proteins that interacted with MHV-68 proteins. The distances between cellular proteins that interacted with MHV-68 proteins in the reference cellular protein interaction network (i.e., the number of protein-protein interactions that must be passed through to connect a pair of proteins) were calculated and plotted. Closed circles (thick line) represent the observed distribution of distances between the MHV-68-interacting proteins. Bars (thin line) show the average distance distribution from an equivalent number of randomly selected cellular proteins. (B) Calculation of a network neighborhood-based priority score. The black circle indicates a cellular protein that interacted with an MHV-68 protein and that served as the starting point in this example. Gray circles indicate other human proteins that interacted with MHV-68 proteins. White circles represent human proteins that did not interact with MHV-68. The priority score was calculated by counting the cellular proteins that interacted with MHV-68 proteins within a distance of four interactions. (C) The priority scores of the top ranked cellular proteins identified in the Y2H screen.
Table 1.
Network properties of cellular proteins that interacted with MHV-68 proteins.
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.
Figure 6.
PCBP1 and TAX1BP1 have opposing effects on MHV-68 replication.
(A) Effect of siRNAs against PCBP1 and TAX1BP1 on MHV-68 replication. Two distinct siRNAs for each gene were tested for their effect on the replication of M3-Luc-MHV-68 as outlined in Fig. 5. Luciferase values were normalized to the level of replication in cells transfected with the negative control siRNA (siGL3). siRTA is a positive control siRNA against the viral RTA. (B) Effect of over-expressing PCBP1 and TAX1BP1 on MHV-68 replication. HEK293T cells were transfected with parental vector pHB (vector), pHB/PCBP1, or pHB/TAX1BP1 and infected with M3-Luc-MHV-68 as in Fig. 5.
Figure 7.
PCBP1 interacts with ORF34 in mammalian cells and negatively regulates late gene expression.
(A) Co-localization of PCBP1 and ORF34 in 3T3 cells. Expression plasmids encoding GFP-tagged PCBP1 and FLAG-tagged ORF34 were co-transfected into NIH 3T3 cells. GFP-PCBP1 was visualized by direct fluorescence. FLAG-ORF34 was detected by immunofluorescence assays with anti-FLAG-M2 antibody and Alexa Fluor 594-conjugated anti-mouse IgG antibodies. Panel 1 shows GFP-tagged PCBP1 (green); panel 2, FLAG-ORF34 (red); panel 3, merged image; panel 4, phase contrast image of same field; panel 5, DAPI-stained nuclei. (B) Co-purification of GST-ORF34 and V5 epitope-tagged PCBP1. GST and GST-ORF34 were expressed in BL21 cells and purified with glutathione beads. Extracts from cells that over-expressed V5-PCBP1 were incubated with either GST or GST-ORF34 bound beads. Lane 1, input amount of V5-PCBP1; lane 2, GST pull-down of V5-PCBP1; lane 3, GST-ORF34 pull-down of V5-PCBP1. (C) Co-purification of GST-PCBP1 and FLAG epitope-tagged ORF34. HEK293T cells were transfected with expression plasmids encoding FLAG-ORF34 and either GST or GST-PCBP1 and then infected with MHV-68. Lysate were incubated with glutathione beads and subjected western blot analysis with anti-FLAG antibodies. Lanes 1 and 3, input amount of FLAG-ORF34; lanes 2 and 4, GST and GST-PCBP1 pull-down of FLAG-ORF34, respectively. (D) Over-expression of PCBP1 did not affect expression from early MHV-68 promoters. Reporter constructs encoding the early viral promoters RTA and ORF57 were co-transfected with parental vector pHB/attR (vector) or pHB/PCBP1 plasmid, with or without the pCMV/RTA plasmid. Luciferase activity was measured at 24 h post transfection. (E) Over-expression of PCBP1 inhibited expression from late MHV-68 promoters. Reporter constructs encoding the late viral promoters M9 and ORF26 were co-transfected with parental vector pHB/attR (vector) or pHB/PCBP1 followed by MHV-68 infection at an MOI of 0.5 in 293T cells. Luciferase activity was measured at 24 h post infection.