Comparative analyses of alphaviral RNA:Protein complexes reveals conserved host-pathogen interactions

Abstract

The identification of host / pathogen interactions is essential to both understanding the molecular biology of infection and developing rational intervention strategies to overcome disease. Alphaviruses, such as Sindbis virus, Chikungunya virus, and Venezuelan Equine Encephalitis virus are medically relevant positive-sense RNA viruses. As such, they must interface with the host machinery to complete their infectious lifecycles. Nonetheless, exhaustive RNA:Protein interaction discovery approaches have not been reported for any alphavirus species. Thus, the breadth and evolutionary conservation of host interactions on alphaviral RNA function remains a critical gap in the field. Herein we describe the application of the Cross-Link Assisted mRNP Purification (CLAMP) strategy to identify conserved alphaviral interactions. Through comparative analyses, conserved alphaviral host / pathogen interactions were identified. Approximately 100 unique host proteins were identified as a result of these analyses. Ontological assessments reveal enriched Molecular Functions and Biological Processes relevant to alphaviral infection. Specifically, as anticipated, Poly (A) RNA Binding proteins are significantly enriched in virus specific CLAMP data sets. Moreover, host proteins involved in the regulation of mRNA stability, proteasome mediated degradation, and a number of 14-3-3 proteins were identified. Importantly, these data expand the understanding of alphaviral host / pathogen interactions by identifying conserved interactants.

Uncovering viral RNA–host cell interactions on a proteome-wide scale

“…Abbreviations: 4SU, 4-thiouridine; ActD, actinomycin D; ChIRP-MS, comprehensive identification of RNA-binding proteins by mass spectrometry; CLAMP, crosslinkassisted messenger ribonucleoprotein purification; Fvo, flavopiridol; HyPR-MS, hybridization purification of RNAprotein complexes followed by mass spectrometry; mRNP, messenger ribonucleoprotein; RAP-MS, RNA antisense purification and quantitative mass spectrometry; SPRI, solid-phase reversible immobilisation; TUX-MS, thiouracil cross-linking mass spectrometry; VIR-CLASP, viral cross-linking and solid-phase purification; vRIC, viral RNA interactome capture. See also [25][26][27][40][41][42][43][44][45][46][50][51][52][53].…”

“…The use of high MOI is not amenable for all viruses and, in some cases, can alter the kinetics of infection [ 99 ]. Other methods, such as vRIC [ 26 ], TUX-MS [ 50 , 51 ], and CLAMP [ 52 , 53 ], combine 4SU RNA labelling with the use of an inhibitor of cellular transcription [actinomycin D (ActD) or flavopiridol (Fvo)] to study the interactions that vRNA establishes with the cell after the replication burst. Because the vRNA-dependent RNA polymerase (RDRP) is insensitive to these compounds, 4SU is mainly incorporated into nascent vRNAs.…”

“…Crosslink-assisted messenger RNP purification (CLAMP) combines formaldehyde crosslinking with another capture strategy, which consists of labelling vRNA with 4SU, followed by in vitro biotinylation of 4SU’s sulfhydryl group with an HPDP-biotin conjugate and purification of the biotinylated vRNA using streptavidin beads. Because biotinylation is performed in cellular extracts, biotin molecules can also bind to the sulfhydryl group present in the cysteine residues within proteins, likely contributing to high false-positive rates ( Box 1 ) [ 52 , 53 ].…”

Alphavirus infection triggers selective cytoplasmic translocation of nuclear RBPs with moonlighting antiviral roles

“…Using this labelled vRNA as a bait, VIR-CLASP revealed that the gRNA of alphaviruses establishes functionally important interactions with hundreds of cellular RBPs during the initial steps of infection (Kim et al, 2020). To profile how these protein-RNA interactions evolve in conditions where viral replication is already active, an alternative method named cross-link-assisted mRNP purification (CLASP) was developed (Gebhart et al, 2020;LaPointe et al, 2018). CLASP employs 4SU labelling of vRNA, followed by formaldehyde crosslinking, 4SU biotinylation and streptavidin purification coupled with proteomics (Gebhart et al, 2020;LaPointe et al, 2018).…”

“…To profile how these protein-RNA interactions evolve in conditions where viral replication is already active, an alternative method named cross-link-assisted mRNP purification (CLASP) was developed (Gebhart et al, 2020;LaPointe et al, 2018). CLASP employs 4SU labelling of vRNA, followed by formaldehyde crosslinking, 4SU biotinylation and streptavidin purification coupled with proteomics (Gebhart et al, 2020;LaPointe et al, 2018).…”