Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Nov 23;95(24):e0059621.
doi: 10.1128/JVI.00596-21. Epub 2021 Sep 29.

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication

Byron Shue  1 , Abhilash I Chiramel  2 , Berati Cerikan  3 , Thu-Hien To  4 , Sonja Frölich  1 , Stephen M Pederson  4 , Emily N Kirby  1 , Nicholas S Eyre  1 , Ralf F W Bartenschlager  3 , Sonja M Best  2 , Michael R Beard  1
Affiliations

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication

Byron Shue et al. J Virol. .

Abstract

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

Keywords: ZIKV; crispr; flavivirus; host factor; organelle; screen; viral replication.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Identification of RACK1 as a critical host factor for the ZIKV life cycle. (A) Schematics of the genome-wide CRISPR/Cas9 screen with ZIKV strategy. (B) Enriched genes after CaRpools analysis isolated several genes of interest including proteins from the EMC complex (EMC1, MMGT1, EMC6) and GNB2L1 (RACK1). (C) Independent polyclonal CRISPR knockouts were performed for EMC1, EMC6 and RACK1, infected with ZIKV (MOI = 2) and qRT-PCR utilised to determine levels of both the respective knockout gene transcripts as well as ZIKV RNA. (D) Plaque assays were also performed concurrently with EMC1, EMC6 and RACK1 knockout cell lines to determine levels of ZIKV virions within the supernatant. All data are *P < 0.05 and n = 3 biological triplicates, representative of similar repeat experiments.
FIG 2
FIG 2
RACK1 is a critical host factor for ZIKV infection. Huh7 cells were transfected with either NTC or RACK1 siRNA and cells harvested for RNA extraction and qRT-PCR (A) or Western blot (B) at indicated time points. (C) HeLa and HTR8 cells were transfected with either NTC or RACK1 siRNA prior to infection with ZIKV (PRVABC59, MOI = 2). 48 hpi, RNA was harvested and qRT-PCR performed. (D) HeLa cells were infected with either ZIKV MR766 or PRVABC59 (MOI = 2) 24 h post NTC/RACK1 siRNA transfection and RNA harvested for qRT-PCR 48 h postinfection. (E) HeLa cells were co-transfected with NTC/RACK1 siRNA with or without plasmid encoding siRNA resistant HA-tagged RACK1. 48 h posttransfection, cells were infected with ZIKV PRVABC59 (MOI = 1) and RNA was harvested for qRT-PCR 24 h postinfection. Prior to infection, WCL was also harvested for immunoblot analysis with mouse RACK1 antibodies. Vinculin served as sample loading control. (F) HeLa cells were infected with PRVABC59 (MOI = 1) 24 h post NTC/RACK1 siRNA transfection and 48 h postinfection, WCL was harvested for immunoblot analysis with chicken flavivirus NS5 and mouse RACK1 antibodies. Vinculin served as sample loading control (G) Immunofluorescence was performed on endogenous RACK1 and ZIKV envelope by transfecting NTC/RACK1 siRNA into HeLa cells and infection with ZIKV (PRVABC59, MOI = 1) 24 h posttransfection. Cells were fixed 48 hpi and stained as indicated (scale bar: 250 μm). All data are *P < 0.05 and n = 3 biological triplicates (except for B and F which are n = 1), representative of similar repeat experiments.
FIG 3
FIG 3
RACK1 is required for flavivirus replication. Huh7 cells (or A549-ACE2 for SARS-CoV-2) were transfected with either NTC/RACK1 siRNA and infected with the following viruses 24 h posttransfection: (A) ZIKV (MOI = 0.5), DENV (MOI = 0.5), WNVNY99 (MOI = 0.5), YFV (MOI = 0.5), LGTV (MOI = 0.5), POWV (MOI = 0.5), (B) SARS-CoV-2 (MOI = 0.1), VSV (MOI = 0.5), EBOV/MAY (103 TCID50) and HSV (MOI = 0.05). Plaque assays were performed to determine viral titers within the supernatant. (C) A549 cells stably expressing the ACE2 receptor was transfected with NTC/RACK1 siRNA prior to infection with SARS-CoV-2 (MOI = 0.1). WCL was harvested and immunoblot analysis performed with primary (anti-vinculin, anti-SARS-CoV-2 spike and anti-RACK1) and secondary (anti-mouse conjugated HRP) antibodies. Vinculin served as sample loading control (n = 3). Data are n = 3 biological triplicates (except for C which is n = 1), representative of similar repeat experiments (*P < 0.05, ns=non-significant).
FIG 4
FIG 4
RACK1 is important prior to flavivirus replication complex formation. (A) Huh7.5 cells were transfected with NTC/RACK1 siRNA for 24 h prior to (B) transfection of DENV2 SGR/SGR GND RNA or (C) infection with DENV2. (B) Cells transfected with DENV-SGR RNA were lysed at the appropriate time points and luciferase assay performed to determine Renilla Luciferase levels. (C) RNA was harvested and extracted from DENV-infected cells 24 hpi and qRT-PCR performed. (D) In contrast, Huh7.5 cells were transfected with DENV2 SGR/SGR GND RNA (E) or infected with DENV2 (F) for 24 h prior to transfection of NTC/RACK1 siRNA for 24 h. (E) Cells transfected with the DENV SGR RNA were lysed at the appropriate time points and luciferase assay performed immediately to determine Renilla Luciferase levels. (F) RNA from DENV infected cells was extracted and qRT-PCR performed (*P < 0.05). Data are n = 3 biological triplicates (except for C which is n = 1), representative of similar repeat experiments (*P < 0.05, ns=non-significant). Data are n = 3 biological triplicates, representative of similar repeat experiments.
FIG 5
FIG 5
RACK1 is critical for the formation of the flavivirus replication complex. (A) Huh7/Lunet-T7 cells were reverse transfected with NTC/RACK1 siRNA for 24 h prior to re-seeding onto glass coverslips. Cells were transfected with pIRO-DENV or -ZIKV constructs for 20 h before fixation and electron microscopy. (B) Immunoblot analysis performed to evaluate RACK1 levels 48 and 72 h posttransfection of siRNA. ß-actin served as sample loading control. (C) Thin-section TEM images of altered morphology of VPs upon transfection of pIRO-DENV or -ZIKV constructs when RACK1 expression is reduced. Upper panel scale bar: 500 nm. Lower panels are magnifications of yellow squared areas in the upper panel images. Lower panel scale bar: 100 nm. (C) For each condition, VPs present within whole-cell sections from 100 cells were counted (E&F) Transfection efficiency of pIRO-DENV and -ZIKV constructs were analyzed via immunofluorescence with anti-NS3 and NS4B primary antibodies and appropriate secondary antibodies and multiple fields of view quantified by counting positive staining cells and normalization to total number of cells. Data are n = 3 biological triplicates (except for C which is n = 1), representative of similar repeat experiments (*P < 0.05).
FIG 6
FIG 6
RACK1 is required for the positioning of flavivirus molecular components during virus replication. Huh7 cells were transfected with NTC/RACK1 siRNA for 24 h prior to infection of DENV2 or ZIKV PRVABC59 (MOI = 1). (A) Cells were subjected to indirect immunofluorescence labeling with primary (anti-NS1 and anti-dsRNA) and secondary (mouse anti-IgG 488 and anti-IgM-555) antibodies. DAPI was used to stain nuclear DNA in the merged images. (B) The signal of both channels was thresholded to remove background noise with green channel designated as region of interest (ROI) and Pearson’s correlation coefficient (PCC) was then determined and plotted as a scatterplot (scale bar: 5 μm, inset scale bar: 2 μm, images representative of n = 3 biological replicates, colocalization was calculated for 10 NTC and 10 RACK1 siRNA treated Huh7.5 cells transfected with either DENV2 or ZIKV PRVABC59).
FIG 7
FIG 7
RACK1 interacts with multiple flavivirus NS1 proteins within the ER lumen. (A) HeLa cells were infected with DENV, WNVKUN or ZIKV (MOI = 1) and 48 hpi, WCL were harvested via sequential incubation with digitonin and NP-40 lysis buffers. Immunoblot analysis was performed with primary (mouse anti-flavivirus NS1 4G4, mouse anti-RACK1 and rabbit anti-Calnexin) and secondary (anti-mouse and rabbit conjugated HRP) antibodies. Vinculin served as sample loading control. (B) Huh7.5 cells were transfected with DENV, WNVKUN, YFV17D or ZIKV NS1-FLAG and RACK1-HA tagged constructs. 24 h posttransfection, cells were fixed and stained with primary antibodies - anti-mouse FLAG/anti-rabbit HA, secondary antibodies – anti-mouse 488/anti rabbit 555. DAPI was used to stain for nuclear DNA (scale bar: 70 μm). (C) HeLa cells were transfected with DENV, WNVKUN, YFV17D or ZIKV NS1-FLAG and RACK1-HA tagged constructs. 24 h posttransfection, cells were fixed and proximity ligation assay performed. DAPI was used to stain for nuclear DNA (scale bar: 15 μm) (D) HeLa cells were infected with DENV2, WNVKUN, YFV17D or ZIKV (MOI = 2) for 24 h and WCL extracted for immunoblot analysis with primary (anti-vinculin, anti-NS1 and anti-RACK1) and secondary (anti-mouse conjugated HRP) antibodies. Vinculin served as sample loading control. Lysates were also used for immunoprecipitation using human anti-NS1 4G4 antibody and subjected to immunoblot analysis with primary (mouse anti-NS1 4G4 and -RACK1) and secondary (anti-mouse conjugated HRP) antibodies. Immunoblot densitometry quantification analysis was performed using Image J for RACK1 levels in flavivirus infected samples relative to mock infection after immunoprecipitation and levels of NS1 relative to loading control (vinculin) prior to immunoprecipitation. Data are n = 3 biological triplicates (except for A and D which is n = 1), representative of similar repeat experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Daep CA, Muñoz-Jordán JL, Eugenin EA. 2014. Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus. J Neurovirol 20:539–560. 10.1007/s13365-014-0285-z. - DOI - PMC - PubMed
    1. Deen J. 2016. The dengue vaccine dilemma: balancing the individual and population risks and benefits. PLoS Med 13:e1002182. 10.1371/journal.pmed.1002182. - DOI - PMC - PubMed
    1. Dawes BE, Smalley CA, Tiner BL, Beasley DW, Milligan GN, Reece LM, Hombach J, Barrett AD. 2016. Research and development of Zika virus vaccines. NPJ Vaccines 1:16007–16007. 10.1038/npjvaccines.2016.7. - DOI - PMC - PubMed
    1. Chatel-Chaix L, Bartenschlager R. 2014. Dengue virus- and hepatitis C virus-induced replication and assembly compartments: the enemy inside–caught in the web. J Virol 88:5907–5911. 10.1128/JVI.03404-13. - DOI - PMC - PubMed
    1. Aktepe TE, Mackenzie JM. 2018. Shaping the flavivirus replication complex: It is curvaceous!. Cell Microbiol 20:e12884. 10.1111/cmi.12884. - DOI - PMC - PubMed

Publication types

MeSH terms

-