Tracking and elucidating alphavirus-host protein interactions.
ABSTRACT Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.
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ABSTRACT: By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification-mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography-MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one's familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2-4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one's data and find the right questions.Nature Protocols 11/2014; 9(11):2539-2554. · 7.78 Impact Factor
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ABSTRACT: Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly-conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.eLife Sciences 06/2014; 3:e02910. · 8.52 Impact Factor
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ABSTRACT: Through an impressive range of dynamic interactions, proteins succeed to carry out the majority of functions in a cell. These temporally and spatially regulated interactions provide the means through which one single protein can perform diverse functions and modulate different cellular pathways. Understanding the identity and nature of these interactions is therefore critical for defining protein functions and their contribution to health and disease processes. Here, we provide an overview of workflows that incorporate immunoaffinity purifications and quantitative mass spectrometry (frequently abbreviated as IP-MS or AP-MS) for characterizing protein-protein interactions. We discuss experimental aspects that should be considered when optimizing the isolation of a protein complex. As the presence of nonspecific associations is a concern in these experiments, we discuss the common sources of nonspecific interactions and present label-free and metabolic labeling mass spectrometry-based methods that can help determine the specificity of interactions. The effective regulation of cellular pathways and the rapid reaction to various environmental stresses rely on the formation of stable, transient, and fast-exchanging protein-protein interactions. While determining the exact nature of an interaction remains challenging, we review cross-linking and metabolic labeling approaches that can help address this important aspect of characterizing protein interactions and macromolecular assemblies.Advances in Experimental Medicine and Biology 01/2014; 806:263-282. · 2.01 Impact Factor