Abstract
Virus–host interactions play vital roles in viral replication and virus-induced pathogenesis. Viruses rely entirely upon host cells to reproduce progeny viruses; however, host factors positively or negatively regulate virus replication by interacting with viral proteins. The elucidation of virus–host protein interaction not only provides a better understanding of the molecular mechanisms by which host cells combat viral infections, but also facilitates the development of new anti-viral therapeutics. Identification of relevant host factors requires techniques that enable comprehensive characterization of virus–host protein interactions. In this study, we developed a proteomic approach to systematically identify human protein kinases that interact potently with viral proteins. For this purpose, we synthesized 412 full-length human protein kinases using the wheat germ cell-free protein synthesis system, and screened them for their association with a virus protein using the amplified luminescent proximity homogenous assay (AlphaScreen). Using this system, we attempted to discover a robust anti-viral host restriction mechanism targeting virus protein X (Vpx) of HIV-2. The screen identified H11/HSPB8 as a Vpx-binding protein that negatively regulates the stability and function of Vpx. Indeed, overexpression of H11/HSPB8 promoted the degradation of Vpx via the ubiquitin–proteasome pathway and inhibited its interaction with SAMHD1, a host restriction factor responsible for blocking replication of HIV. Conversely, targeted knockdown of H11/HSPB8 in human trophoblast cells, which ordinarily express high levels of this protein, restored the expression and function of Vpx, making the cells highly susceptible to viral replication. These results demonstrate that our proteomic approach represents a powerful tool for revealing virus–host interaction not yet identified by conventional methods. Furthermore, we showed that H11/HSPB8 could be a potential host regulatory factor that may prevent placental infection of HIV-2 during pregnancy.
Highlights
To replicate and propagate, viruses utilize sophisticated mechanisms to hijack the machinery and materials of their host cells
When a relative light unit per cutoff (RLU/Co) ratio of ≥2.25 was used as the threshold, we found 15 host kinases that could selectively interact with HIV-2 virus protein X (Vpx)
Identification of new virus–host cell interactions is a key challenge in clarifying the nature of viral infection and pathogenesis
Summary
Viruses utilize sophisticated mechanisms to hijack the machinery and materials of their host cells. In a virally infected cell, host proteins play crucial roles in multiple biological processes that promote viral replication by providing molecular architecture or functional assistance. To this end, viral proteins need to interact directly or indirectly with host cell proteins that are relevant to the viral life cycle. Some host proteins operate as anti-viral factors to counteract or restrict viral replication within infected cells. Certain viral proteins counteract these anti-viral proteins, resulting in sustained viral propagation. A comprehensive understanding of dynamic host–virus protein interaction would greatly improve our understanding of the viral life cycle and pathogenesis
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