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Interacting Partners of CHIKV nsP3 in Human Macrophages

ABSTRACT: Chikungunya virus (CHIKV), an alphavirus transmitted by mosquitoes, has instigated several epidemics in recent years, sparking intensive efforts to understand its biology. Despite progress, the understanding of CHIKV’s molecular interactions with host cell constituents, especially in susceptible cells such as macrophages remain limited. We used mass spectrometry platform to characterize the interactions between CHIKV-nsP3, a viral nonstructural protein, and proteins in human THP-1 macrophage cells. Our findings revealed 196 high-confidence interactions primarily involving nsP3. Further the sub-cellular localization, pathways these interacting proteins might be involved in were deduced using computational methods. The interacting partners were further incorporated into a comprehensive host virus interaction network derived from extensive literature on alphavirus-host interactions. Collectively, this study offers the first interaction map between CHIKV nsP3 protein and THP-1 cells, illuminating new probable roles of host cell proteins in CHIKV’s replication cycle.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sujatha Sunil

LAB HEAD: Dr. Sujatha

PROVIDER: PXD045252 | Pride | 2024-03-06

REPOSITORIES: Pride

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Project description:Pooled CRISPR knock-out (KO) screens using live viruses are a proven and valuable approach to identify essential host factors acting across the viral life cycle. Here we describe the development of a pooled, genome-wide CRISPR KO screening approach using stable viral replicon cell lines to specifically target host genes essential for viral replication complex formation and function. We benchmarked this approach with dengue virus 2 (DENV2), a model virus with well-established replicon systems and knowledge of the host factors requirements for replication. We developed a stable fluorescent DENV2 replicon cell line to perform a pooled genome wide FACS-based CRISPR KO screen. Our benchmark DENV2 screen successfully identified previously known host factors involved in DENV replication such as components of the ER membrane complex (EMC) and oligosaccharyltransferase (OST) complex. In addition, 2 other genes identified in the screen (DOHH and ZFP36L2) were validated to impact DENV2 replication. We extended the replicon screening approach to two different classes of viruses - Chikungunya virus (CHIKV), a positive sense RNA virus that is known to replicate at the plasma membrane, and Zaire Ebola Virus (ZEBOV), a negative sense RNA virus that replicates in cytoplasmic inclusion bodies. The CHIKV replicon screen identified several important host factors involved in CHIKV replication, including CSDE1 and GOLGA7 that were successfully validated in the context of live CHIKV infection. A distinct set of host factors such as Euchromatic histone-lysine N-methyltransferase 1 and 2 (EHMT1, 2), and Ubiquitin specific peptidase 7 (USP7) were identified in the ZEBOV replicon screen and were further validated using independent transient transfection assays. Comparative analysis of the combined hits from all 3 screens reveals distinct subsets of host pathway requirements for each virus. Thus, viral replicons-based screens can be a useful tool that can be extended to diverse classes of viruses to identify host pathways essential for viral replication and to uncover potential novel targets for host-directed antiviral therapeutics.

			Action	DRS
	H1.raw	Raw
	H2.raw	Raw
	H3.raw	Raw
	ICGEB-HUMAN-UNIPROT.pdResult	Other
	c1.raw	Raw

Dataset Information

Interacting Partners of CHIKV nsP3 in Human Macrophages

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