Multiple Routes to Oncogenesis Are Promoted by the Human Papillomavirus-Host Protein Network.

We have mapped a global network of virus-host protein interactions by purification of the complete set of human papillomavirus (HPV) proteins in multiple cell lines followed by mass spectrometry analysis. Integration of this map with tumor genome atlases shows that the virus targets human proteins frequently mutated in HPV- but not HPV+ cancers, providing a unique opportunity to identify novel oncogenic events phenocopied by HPV infection. For example, we find that the NRF2 transcriptional pathway, which protects against oxidative stress, is activated by interaction of the NRF2 regulator KEAP1 with the viral protein E1. We also demonstrate that the L2 HPV protein physically interacts with the RNF20/40 histone ubiquitination complex and promotes tumor cell invasion in an RNF20/40-dependent manner. This combined proteomic and genetic approach provides a systematic means to study the cellular mechanisms hijacked by virally induced cancers.Significance: In this study, we created a protein-protein interaction network between HPV and human proteins. An integrative analysis of this network and 800 tumor mutation profiles identifies multiple oncogenesis pathways promoted by HPV interactions that phenocopy recurrent mutations in cancer, yielding an expanded definition of HPV oncogenic roles. Cancer Discov; 8(11); 1474-89. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.

Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity.

HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFß, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFß is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

Quantitative analysis of triple-mutant genetic interactions.

The quantitative analysis of genetic interactions between pairs of gene mutations has proven to be effective for characterizing cellular functions, but it can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed triple-mutant analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, which is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principal actors are deleted. TMA has also uncovered double-mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete and measures interactions for up to 30 double mutants against a library of 1,536 single mutants.

Systematic triple-mutant analysis uncovers functional connectivity between pathways involved in chromosome regulation.

Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA). We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1Δ cac1Δ to TMA, we found that the Swi/Snf Rdh54 protein compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1Δ strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered through traditional double-mutant analyses.