Figure 1.
Intraviral protein interactions in HSV-1.
(A) Phylogeny of the five investigated herpesviruses and their classification into the three subfamilies α, β, and γ. A timeline is added to indicate approximately when the different subfamilies were separated based on findings by McGeoch and colleagues [6],[7]. (B) Intraviral protein-protein interaction network for HSV-1. The proteins are coloured according to their conservation in the herpesvirus phylogeny: the blue nodes are core proteins conserved in all five viruses, two nodes (pink) are conserved in α and γ herpesviruses, several red ones in α herpesviruses and the grey ones are specific to HSV-1. Edges indicate observed interactions in HSV-1, and red edges indicate previously reported interactions. The protein interaction network was generated using the Cytoscape software (www.cytoscape.org) [58].. (C) node degree distribution on a linear or logarithmic (inset) scale. The herpesviral networks can be approximated by power law distributions (Table S3). (D) Simulations of deliberate attack on HSV-1 in comparison to two human networks by removing their most highly connected nodes in decreasing order. After each node is removed, the new network characteristic path length (average distance between any two nodes) of the remaining network is plotted as a multiple or fraction of the original parameters. The herpesviral networks consistently exhibited a higher attack tolerance, as the increase in path length is considerably smaller.
Figure 2.
Overlap of herpesviral protein-protein interaction networks.
(A) shows the sunflower structure induced by the protein sets of the five viruses and their intersections. For each overlapping area the number of shared proteins with detected interactions, in addition to the total number of shared proteins are indicated. All shared proteins in the various subgroups are interacting (with the exception of the β+γ subgroup where only three out of six shared proteins are interacting) while for the individual viruses between 50% and 70% of the proteins have observed interactions. (B) Comparison of conserved interactions between orthologous proteins for any two herpesvirus species. In each rectangle, the value above the lines indicates the observed number of homologous interactions detected in both herpesviruses (in green). The value below the line (in black) gives the total number of interactions detected in the first species (indicated in columns) between proteins which have orthologs in the second species (indicated in rows). On the diagonal, the total number of interactions is shown for each virus. (C) Distribution of the number of conserved interactions between HSV-1 and mCMV for 1000 random orthology assignments (blue line) in comparison to the true number of conserved interactions (red vertical line). For each pairwise comparison, subnetworks were selected between proteins conserved in both viruses and then the orthology assignments between the proteins were randomized. Accordingly, the size and degree distribution of the subnetworks does not change. (D) Comparison of the number of interactions conserved in 2, 3, 4 and 5 species for 1000 random orthology assignments (yellow boxes) to the true number of interactions conserved in that many viruses (red line). Random orthology assignments were created in a similar way as for Figure 2C.
Figure 3.
The core network in herpesviruses.
(A) Core interaction network between the 41 core orthologous proteins. The width of the edges indicates the number of species in which the interaction was detected. Proteins are labelled with the HSV-1 protein names. Networks were illustrated using Cytoscape [58]. (B) The left plot of the figure shows the number of species for which an interaction is observed (y-axis), plotted against the sequence similarity of the two interacting proteins (x-axis). No correlation between sequence similarity and number of conserved interactions was detected. This is further quantified in (C) showing the distribution of similarities of the interacting proteins as compared to the background distribution of similarities derived from sequence similarities of protein pairs not interacting. (D) Compares the distribution of interactions conserved in two species within or across subfamilies against the random expectation if all possible combinations are equally likely or weighted based on the number of interactions in the core of each species. Interactions are not conserved preferentially between closely related species and no significant difference to the random expectation can be observed. (E) Phylogenetic tree for the herpesviral core network. Bootstrap values are indicated on the internal nodes.
Figure 4.
The non-core network in herpesviruses.
(A) Interaction networks of the non-core proteins of the five herpesviruses. All core proteins and the interactions between them are reduced to one central node (the core) surrounded by the leaves formed by the subgroup and individual interaction networks. The colour code is the same as in Figure 1B. (B) Phylogenetic tree based on the complete herpesvirus protein networks. Bootstrap values are indicated on the internal nodes.
Figure 5.
Core interactions are conserved.
(A) Histogram indicating the percentage of interactions confirmed either by Y2H, CoIP or Y2H and CoIP. We tested in total 92 interactions in HSV-1, mCMV and EBV predicted from 55 interactions detected in KSHV between proteins conserved in at least one other species. For all species more than 50% of the predicted interactions could be confirmed by CoIP, while the numbers confirmed by Y2H were significantly lower. (B) Illustrates how the discovery of new interactions decreases for each new interactome. Average number of new interactions found after 1, 2, 3, 4 and 5 Y2H screens (red points) is plotted, where averages are taken over all possible sequences of Y2H screens. The green line shows a linear best fit line. While there is a clear decrease in the number of new interactions discovered for each additional Y2H analysis, there seems to be a significant number of interactions still to be found.
Figure 6.
High-confidence protein interactions in herpesvirus virions.
(A) Schematic map of protein interactions within herpesvirus virions. The viral proteins indicated (or their orthologs) have been shown to be present in virus particles by proteomic analyses [59],[60]. The central nodes indicate capsid proteins, the middle layer tegument proteins and the outer layer glycoproteins. The colour code is the same as in Figure 1B. Indicated are all interactions detected in one of the five species and the width of the edges indicates the number of species in which the interactions were detected. M51 is marked by the red arrow. (B) mCMV M50, M51 and M53 co-localize at the nuclear membrane. CFP-tagged M50, YFP-tagged M51 and DsRed2-tagged M53 were expressed either alone (upper row) or together (lower 2 rows) and analysed by fluorescence or bright field (BF) microscopy.