An integrated map of HIV-human protein complexes that facilitate viral infection

Abstract

Recent proteomic and genetic studies have aimed to identify a complete network of interactions between HIV and human proteins and genes. This HIV-human interaction network provides invaluable information as to how HIV exploits the host machinery and can be used as a starting point for further functional analyses. We integrated this network with complementary datasets of protein function and interaction to nominate human protein complexes with likely roles in viral infection. Based on our approach we identified a global map of 40 HIV-human protein complexes with putative roles in HIV infection, some of which are involved in DNA replication and repair, transcription, translation, and cytoskeletal regulation. Targeted RNAi screens were used to validate several proteins and complexes for functional impact on viral infection. Thus, our HIV-human protein complex map provides a significant resource of potential HIV-host interactions for further study.

Figure 1. Workflow Overview.

HIV-interacting proteins and RNAi phenotypes are mapped to a network of human protein functional interactions (yellow and red nodes respectively). Network propagation is performed separately for each of these two mappings. Significant genes are selected based on the combination of both propagation results (blue nodes). Finally, enriched HIV-human protein complexes are identified within the list of significant genes (HIV proteins added as green nodes, protein complexes highlighted by circles).

Figure 2. Predictive power and statistical results.

(A) Pearson correlation of RNAi and APMS network propagation scores (green dot). The green line shows the density plot of random correlation coefficients based on permuting the relationship between network nodes and protein names. Note that random correlation is not zero due to the network structure of HumanNet which is not randomized. (B) The RNAi and APMS network propagation scores for each protein. Blue dots are proteins significant in both propagations. (C) ROC curve showing the predictive power of RNAi-propagation (blue) and APMS-propagation (red).

Figure 3. Validation of mRNA knock-down by siRNAs found to alter HIV infection.

293T cells were transfected with siRNA against the identified genes and two non-targeting scramble siRNAs. 72 h post-transfection, total RNA was harvested and used to make a cDNA library. The presence of the target gene and a housekeeping gene, TBP, was measured using QPCR. Target gene levels were normalized to TBP within in each sample. Values reported are normalized target gene levels compared to values observed in transfections with non-targeting scramble siRNAs.

Figure 4. Map of HIV-human protein complexes.

40 identified human protein complexes are shown together with the HIV protein targeting the complex. Green rectangles correspond to HIV proteins. Human complexes are shown as ellipses. A color gradient from red (high) to yellow (low) indicates the average rank of the complex in the APMS- and RNAi-propagations. Node size corresponds to number of subunits in the complex. Gray edges represent functional interactions between the human complexes; green edges are HIV-human interactions. Purple boxes indicate protein complexes that were selected for follow-up RNAi screens.

Figure 5. Selected complexes and RNAi screening results.

(A) Profilin-1 complex interacting with GP160. (B) DNA-PK-Ku-eIF2-NF90-NF20 complex interacting with NC. (C) LARC complex interacting with Gag. Interactions within the complex represent functional interactions from HumanNet (green), manually curated interactions from the Metabase resource (gray) or from both sources (red). Pink vs. turquoise stars correspond to proteins that were confirmed in our RNAi validation screen vs. previous screens, respectively. Orange nodes are kinases, red transcription factors, blue are binding proteins as classified in Metabase. The bar plots show the HIV luciferase activity of the sample normalized by the HIV luciferase activity of control siRNAs. (D) HIV luciferase activity for three non-targeting siRNAs (positive controls) and luciferase-targeting siGL3 (negative control) performed simultaneously with siRNA transfections shown in A, B, and C.