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. 2024 May 3;16(5):725.
doi: 10.3390/v16050725.

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication

Shwetha Shivaprasad  1   2 , Wenjie Qiao  1 , Kuo-Feng Weng  1 , Pavithra Umashankar  2 , Jan E Carette  1 , Peter Sarnow  1
Affiliations

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication

Shwetha Shivaprasad et al. Viruses. .

Abstract

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

Keywords: CRISPR screen; dengue virus; host–virus interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bubble plot of results from the DENV CRISPR mini-pool library screen. (A) Schematic for the CRIPSR mini-pool library screen in Huh7 cells. (B) Candidate genes identified by the CRISPR-Cas9 screen. Data analysis was performed using the MAGeCK algorithm to identify enriched sgRNA, and the genes were ranked by robust rank aggregation (RRA) scores. (C) Volcano plot displaying the log2 fold change and FDR for genes identified in the screen. Genes with a threshold of FDR < 0.01 and log2 fold change > ±1 are labeled.
Figure 2
Figure 2
Effect of PACT knockout on DENV replication. (A) Western blot depicting depletion of PACT in Huh7 cells transduced with lentiviruses expressing sgRNA#1, sgRNA#2, or sgRNA#3 targeting the PACT gene. (B) Huh7 wildtype and PACT knockout cells were infected with DENV2-Luc reporter virus at an MOI of 0.1, and cells were harvested 48 h post-infection for measurement of luciferase activity. Luciferase expression in cell lysates is plotted as an average from three independent experiments (n = 3, **** p < 0.0001). (C) A clonal PACT knockout Huh7 cell line expressing sgRNA#3 was generated by cell sorting and infected with DENV2-Luc reporter virus at an MOI of 0.1. Infected cells were either untreated or treated with the viral replication inhibitor MK-0608. Luciferase expression was measured at the indicated time points and plotted as an average from three independent experiments (n = 3, **** p < 0.0001). (D) Western blot for DENV NS3 and DENV NS4B in Huh7 wildtypes and PACT knockout cells infected with DENV2 16681 at the indicated MOI. Densitometry values are indicated below the respective lanes. (E) Huh7 wildtype and PACT knockout cells were transfected with either DENV-RLuc wildtype replicon RNA or (F) DENV-RLuc replication defective (NS5-GDD) RNA together with a Firefly luciferase (FLuc)-expressing control mRNA. Cells were harvested at the indicated time points in six independent replicates, and relative luciferase expression is plotted as the ratio of Renilla and Firefly luciferase activities for both wildtype and replication-defective DENV RNAs (**** p < 0.0001).
Figure 3
Figure 3
Effect of PACT knockout on distinct viral infections. (A) Huh7 wildtype and PACT knockout cells were infected with DENV-1, DENV-2, DENV-3, or DENV-4 at an MOI of 0.1, and viral RNA abundances were measured by qPCR using universal DENV primers. Data are represented as the average fold change over wildtype cells from three independent experiments (** p < 0.01, * p < 0.1). (B) Huh7 wildtype and PACT knockout cells were infected with Dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV), or chikungunya virus (CHIKV) at an MOI of 0.1 and harvested 48 h post-infection. Viral RNA abundances were measured by qPCR using specific primers. Data are represented as average fold-change over wildtype cells (**** p < 0.0001, ** p < 0.01).
Figure 4
Figure 4
Effect of ectopic expression of PACT on DENV infection in HEK293/FH-PACT cells. (A) HEK293/FH-PACT cells were cultured in the presence or absence of doxycycline and infected with the DENV2-Luc reporter virus at an MOI of 0.1. Luciferase expression was measured from cells harvested at the indicated time points post-infection and plotted as an average of six independent replicates (n = 3, **** p < 0.0001). (B) Western blot for PACT protein expression and (C) luciferase expression from DENV2-Luc reporter virus-infected wildtype, mCherry knock-in, and myc-PACT knock-in HEK293/FH-PACT cells, cultured in the presence or absence of doxycycline (**** p < 0.0001). (D) Domain organization of PACT and its RRM deletion mutants. (E) HEK293/FH-PACT cells expressing wildtype or truncated versions of PACT were cultured in the presence or absence of doxycycline and infected with the DENV2 luciferase reporter virus at an MOI of 0.1. Luciferase expression was measured 72 h post-infection and plotted as an average of six independent replicates (**** p < 0.0001, ** p < 0.01).
Figure 5
Figure 5
Gene expression data. Differentially expressed genes in uninfected and DENV1 (MOI 0.1) infected (A) Huh7 wildtype or (B) PACT clonal knockout cells at 48 h post-infection were visualized using volcano plots. ER stress response genes that were more than 2-fold upregulated are labeled. GO enriched genes for each condition are depicted in bar plots.
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