Deciphering flavivirus–host interactions using quantitative proteomics
Introduction
Flaviviruses are a large group of medically relevant viruses that cause significant disease in humans and animals. These include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) [1,2]. Flavivirus virions contain the structural proteins, an envelope as well as a positive-sense, single-stranded RNA genome of approximately 11 kb in length. The incoming flavivirus genome encodes a single open reading frame that is translated on the endoplasmic reticulum (ER) into a single polyprotein. This polyprotein is subsequently cleaved into three structural proteins (capsid, prM and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by viral and host proteases. The structural proteins form the viral particles, while the non-structural proteins are required for intracellular viral propagation and immune evasion [1,2].
Given their limited protein repertoire, flaviviruses rely on the host cell machinery for many steps in their life cycle. Identification of host proteins that are required for viral replication can inform the development of effective host-directed therapeutics and new innate antiviral mechanisms. Recent advances in mass spectrometry (MS)-based proteomics have increased the sensitivity and specificity and allow for the systematic identification and quantification of proteins in a high-throughput manner (Box 1) [3]. This technology is being widely used in many fields, and has been used to identify host proteins that are involved in viral replication including flaviviral infection (Figure 1). Here, we review how proteomic approaches have improved our understanding of flavivirus–host interactions, highlighting the mechanisms by which flaviviruses manipulate host cellular processes to promote infection.
Section snippets
Mapping virus–host protein–protein interactions (PPIs)
Viruses hijack host machinery to ensure efficient viral replication. This is often achieved via physical interactions between viral and host proteins. By employing affinity purification-mass spectrometry (AP-MS) coupled with RNA interference (RNAi) screening, Li et al. generated WNV-host PPIs map and identified 26 virus-interacting host proteins that impact WNV infection [4••]. In particular, it was shown that WNV capsid interacts with PYM1, a host protein involved in the exon-junction complex
Identifying the interactomes of individual viral proteins
While some groups have taken a broad approach to identify a comprehensive flavivirus–host interactome, others have defined the interactors of an individual viral protein critical for infection. The non-structural proteins of flaviviruses play diverse roles in viral replication and assembly and in antagonizing the host immune response, including type I IFN signaling and RNA interference (RNAi). The flaviviral non-structural protein, NS2A, was shown to suppress RNAi in both mammals and
Identifying host proteins that associate with viral RNA
By performing comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), Ooi et al. identified 464 host proteins that interact with DENV or ZIKV genomic RNA [32••]. Complementary CRISPR and haploid genetic screens with multiple clinical isolates of DENV and ZIKV revealed overlap between these approaches. Indeed, a subset of ER-associated proteins that play an important role during viral infection including RRBP1 and vigilin, were shown to bind to the viral genomic
Detecting host protein dysregulation upon viral infection
While we have a deep understanding of the changes in the gene expression landscape during infection, we have a much poorer understanding of how the proteome changes. Several groups have employed MS to define the changes in host protein abundance during flaviviral infection. Dong et al. used MS to quantify differential regulation of host proteins during DENV infection in 293T cells and found that DDX21 levels are decreased in infected cells. Upon infection, DDX21 translocates from the nucleus to
Discovering post-translational modifications of host proteins in virus-infected cells
Post-translational modifications (PTMs) can regulate protein function, localization and stability. One strategy utilized by viruses to manipulate host proteins is to alter PTMs. Protein ubiquitylation is one of the most common PTMs that can play important roles in regulating protein stability and function. Following immunoprecipitation and MS analysis, Zhang et al. identified host proteins that are differentially ubiquitylated in DENV-infected cells [58•]. AUP1, a lipid droplet-localized
Concluding remarks
The interplay between viruses and their hosts is complex. To investigate such interactions, it is important to obtain a comprehensive view of both viral and host proteins during infection, and not just at the RNA level. MS-based proteomics approaches can be used to quantify protein abundance, interactions, and PTMs upon viral infection and provide researchers with opportunities to better understand the mechanisms by which viral infection alters the host cellular machinery.
AP-MS is a robust
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by grants from the National Institutes of Health to S.C. (5R01AI122749, 1R01AI140539, 1R01AI150246) and H.R. (1RO1AI143850). S.C. is a recipient of the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Award. We apologize to all colleagues whose contributions were not cited due to space limitations.
References (61)
- et al.
Flavivirus induces and antagonizes antiviral RNA interference in both mammals and mosquitoes
Sci Adv
(2020) - et al.
Dengue virus hijacks a noncanonical oxidoreductase function of a cellular oligosaccharyltransferase complex
mBio
(2017) - et al.
Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells
Virology
(2015) - et al.
Global interactomics uncovers extensive organellar targeting by Zika virus
Mol Cell Proteomics
(2018) - et al.
Flavivirus infection impairs peroxisome biogenesis and early antiviral signaling
J Virol
(2015) - et al.
The dengue virus NS5 Protein intrudes in the cellular spliceosome and modulates splicing
PLoS Pathog
(2016) - et al.
Development of viable TAP-tagged dengue virus for investigation of host–virus interactions in viral replication
J Gen Virol
(2016) - et al.
Identification of RNA binding proteins associated with dengue virus RNA in Infected cells reveals temporally distinct host factor requirements
PLoS Negl Trop Dis
(2016) - et al.
Annexin II as a dengue virus serotype 2 interacting protein mediating virus interaction on vero cells
Viruses
(2019) - et al.
A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication
Nat Microbiol
(2018)