Global mapping of herpesvirus-host protein complexes reveals a transcription strategy for late genes

Abstract

Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi's sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers, and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle.

Figure 1. Assembly and broad characterization of the KSHV-human interactome

(A) Summary of the workflow used for assembly of the PPI network. (B) The MiST feature weights and score threshold were subjected to an exhaustive parameter grid search and the optimal values were empirically determined by maximizing the area under the curve defined by the True Positive versus False Positive rate. The depicted receiver operating characteristic (ROC) curve illustrates MiST prediction accuracy with optimal feature weights for specificity (0.5) and reproducibility (0.5) with the curve inflection point marked with an x. The ROC curve was used to help define an appropriate MiST threshold of 0.7. This corresponded to 564 high confidence interactions (see also Tables S1 and S2). (C) Venn diagram showing the overlap between previously reported KSHV-host interactions and the high confidence PPI network. The bar graph shows the number of host proteins associating with each viral protein, ranked in descending order, with the previously reported interactions noted in red (see also Table S2). (D–E) Host genes that interact with viral proteins unique to KSHV show significantly elevated signs of recent selection. Shown are the distributions of mean differences from background across 10,000 bootstrap samples. Signatures of selection are measured using a mammalian based dN/dS statistic for ancient selection (D), and the iHS statistic based on the 1000 Genomes Project for recent selection (E). (F–G) Bar graphs showing the percentage of all human proteins, versus the subset that interact with KSHV lytic or latent proteins, that are associated with cancer (F) or non-cancer diseases (G) in the DisGeNET database. * Indicates a p-value < 0.05 and ** indicates a p-value < 0.005 as determined by the hyper-geometric test (see also Figure S1 and Table S3). (H) Venn diagram depicting the overlap between the high confidence HEK293T interaction partners of KSHV and HIV-1. The interaction partners are broken out into the number of cellular proteins in each category. The p-value was determined by the hyper-geometric test (see also Table S4).

Figure 2. Network representation of the KSHV-host interactome

The full, high confidence interaction network contains 67 KSHV proteins (gold nodes) and 556 cellular proteins (blue nodes). Black edges indicate known interactions between cellular proteins associated with each individual viral protein. A subset of the identified complexes is labeled. NADH UOR: ubiquinone oxidoreductase; TFIIIC: transcription factor for polymerase III C; SSB repair: single-stranded break repair; 5FMC: 5 friends of methylated Chtop; CDK: cyclin dependent kinase; RSKs: ribosomal s6 kinases; IKK: IκB kinase complex; RPA: replication protein A complex; RNA Pol II: RNA polymerase II; CPSF: cleavage and polyadenylation specificity factor complex; APC: anaphase-promoting complex; CTLH: C-terminal to LisH motif complex (see also Figure S2 and Table S5).

Figure 3. Using the KSHV interactome to predict viral protein functions

Summary of predicted functions of KSHV proteins, derived from GO term and protein domain enrichments within the set of high confidence PPIs associated with each viral protein (see also Figure S3 and Tables S6 and S7).

Figure 4. KSHV ORF24 binds human RNA polymerase II to drive viral late gene expression

(A) Network showing the high confidence interaction partners of ORF24 identified in 293T cells (all blue nodes), and those also identified in KSHV infected iSLK.219 cells (blue nodes with heavy black borders). Pol II subunits are depicted to the left of the ORF24 node while Pol II accessory factors are shown to the right. Grey lines indicate known interactions between human proteins. (B) Lysates of iSLK cells either latently (− doxycycline) or lytically (+ doxycycline) infected with KSHV.FLAG.24 were subjected to α-FLAG IP and Western blotted for the RPB1 subunit of Pol II. GAPDH was used as a loading and IP specificity control. In this and all subsequent IPs, input represents 5% of lysate used for IP. (C) Lytically induced iSLK cells infected with KSHV.WT were transduced with FLAG-tagged ORF24, and lysates were subjected to α-FLAG IP and Western blotted using antibodies against total RPB1 (Pol II) or the hyperphosphorylated forms of Pol II. (D) Heat map representing the MiST scores for Pol II subunits detected in association with the ORF24 orthologs from KSHV (ORF24), MHV68 (mORF24), EBV (BcRF1), and CMV (UL87) via AP-MS from 293T cells. (E) 293T cells were transfected with plasmids expressing FLAG-tagged full length (FL) ORF24 or fragments of ORF24 encompassing the N-terminus (aa 1-401), central domain (aa 402-603), or C-terminus (aa 604-752). Cell lysates were subjected to α-FLAG IP and Western blotted for the RPB1 subunit of Pol II. (F) A Clustal Omega multiple sequence alignment for the ORF24 orthologs showing the stretch of 5 conserved residues within their N-termini. (G) 293T cells were transfected with plasmids expressing FLAG-tagged WT ORF24 or an ORF24 mutant in which the conserved ‘RLLLG’ motif was mutated to ‘RAAAG’ or ‘AAAAA’. Lysates were subjected to α-FLAG IP and Western blotted with the indicated antibodies. (H) Heat map representing the MiST scores for Pol II subunits detected in association with WT ORF24 (ORF24) versus the ORF24^RAAAG mutant via AP-MS from 293T cells. (I–J) iSLK cells infected with WT KSHV or KSHV lacking ORF24 (KSHV.24.Stop) were transduced with a retroviral vector expressing either WT ORF24 or the indicated ORF24 Pol II binding mutants. Forty-eight hr following lytic reactivation, accumulation of the ORF52 late gene (I) or the ORF38 early gene (J) was measured by RT-qPCR and normalized to levels of 18S, with the viral mRNA levels present during WT infection set to 1. * Indicates a p-value < 0.05 and ** indicates a p-value < 0.005 as determined by Student’s t-test (see also Figure S4).

Figure 5. ORF24 binds late gene promoter DNA in a manner essential for late gene expression

(A) Purified WT ORF24 or ORF24 putative DNA-binding mutants (ORF24^{N425A, N427A} and ORF24^{N518A, N520A}) (0–280 nM) were subjected to EMSA with 23 nt radiolabeled dsDNA probes encompassing the K8.1 late promoter, which contains a central ‘TATT’ sequence, or the ORF57 early promoter, which contains a central ‘TATA’ sequence. Where indicated, the ‘TATT’ motif in the K8.1 promoter probe was mutated to ‘CCCC’. The specificity of the interaction was confirmed by competition with unlabeled (cold) probe. (B) 293T cells were transfected with plasmids expressing strep-tagged WT ORF24 or the indicated ORF24 DNA-binding mutants. Lysates were subjected to α-strep IP and Western blotted for the RPB1 subunit of Pol II. Tubulin was used as a loading and IP specificity control. (C–D) iSLK cells infected with WT KSHV or KSHV.24.Stop were transduced with a retroviral vector expressing WT ORF24 or the indicated ORF24 DNA-binding mutants. Forty-eight hr following lytic reactivation, accumulation of the ORF52 late gene (C) or the ORF38 early gene (D) was measured by RT-qPCR and normalized to levels of 18S, with the viral mRNA levels present during WT infection set to 1. * Indicates a p-value < 0.05 as determined by Student’s t-test (see also Figure S5).

Figure 6. ORF24 functionally replaces TBP on the late promoter

(A–B) Chromatin from iSLK cells lytically infected with KSHV.FLAG.24 was isolated and subjected to ChIP using antibodies against FLAG (to precipitate FLAG ORF24), TBP, and Pol II (A), or TFIIB and TFIIH (ERCC3) (B). Co-precipitating DNA was detected by PCR using radiolabeled nucleotides and primers specific for the indicated promoter or gene body. Experiments were repeated at least three times and a representative image is presented with the ChIPs quantified against input samples (see also Figure S6).

Figure 7. Model for ORF24 activation of late gene promoters

ORF24 interacts with Pol II through conserved leucines in its N-terminal domain. The polymerase is then brought to the late promoter TATT box via the ORF24 TBP-like domain. TBP is excluded from late promoters, and instead ORF24 binding nucleates assembly of the PIC by enabling recruitment of other cellular (GTFs).