Tracking and elucidating alphavirus host protein interactions
CHIKV has a positive-sense, single-stranded RNA genome (g RNA) that encodes two polyproteins.
Immunofluorescence was performed on methanol fixed He La cells for detection of BNIP3 using Anti-BNIP3 (8HCLC) Recombinant Rabbit Oligoclonal Antibody (Product# 710728, 2ug/ml), alpha-Tubulin Monoclonal Antibody (Product# 322500, 1ug/ml) and labeled with Goat anti-Rabbit Ig G (H L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 00), Goat anti-Mouse Ig G Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A11032, 0) respectively.
Replication of incoming positive-sense genomic RNA, which occurs in membrane-associated complexes containing the viral nonstructural proteins (ns Ps), first generates a minus strand copy, followed by new progeny plus strand RNAs.
We hypothesize that in addition to known differences in the viral ns P components of the minus and plus strand replicases, that recruitment of different host factors facilitates their disparate functions.
This rapid purification helps preserve protein complexes close to their original state in the cell and minimizes nonspecific interactions.
Given the wide use and availability of GFP-tagged protein reagents, the present method should greatly facilitate the elucidation of many cellular processes.
Protein complexes mediate the majority of cellular processes.
Knowledge of the localization and composition of such complexes provides key insights into their functions.
Here we describe the first known function of ns P3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci.
Panel a) shows representative cells that were stained for detection and localization of BNIP3 protein (green), Panel b) is stained for nuclei (blue) using Slow Fade® Gold Antifade Mountant with DAPI (Product # S36938,).
Panel c) represents cytoskeletal alpha-tubulin staining (red).
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