Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation

Abstract

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.

Keywords: GARP complex; HAP1 screen; poxviruses; retrograde transport.

FIG 1

Haploid genetic screen identifies host factors required for MPXV infection in HAP1 cells. (A) Significance of enrichment of gene-trap insertions for MPXV ROC. (B) Significance of enrichment of gene-trap insertions for MPXV WA. Each circle represents a gene, and the size of the circle represents the number of independent gene-trap events identified in the selected resistant population. The y axis shows the significance of enrichments of gene-trap insertions between selected and unselected cell populations calculated using Fisher's exact test. The top 20 genes with the highest significance are labeled according to the functional annotation group.

FIG 2

Functional gene annotation analyses of significant MPXV host factors. (A) MPXV ROC and WA enriched genes with an adjusted P value of <0.05. Genes listed in the middle were identified in both screens. (B) Overview of functional annotation ClueGO analysis. Similar terms were grouped into one representative term. The group sections represent the relative numbers of terms in the groups. The significance value for each group is indicated. **, P ≤ 0.001.

FIG 3

GARP complex genes are required for MPXV EV production. (A and B) HEK293FF6 cell lines containing VPS WT, VPS51KO, VPS52KO, VPS53KO, or VPS54KO genes were infected with MPXV ROC (A) or WA (B) at a multiplicity of infection (MOI) of 1 in the absence and presence of 2 μM ST-246. MV and EV production was measured 24 h postinfection (hpi) in each cell line. The percentage of EV released was determined by calculating the proportion of EV represented in the total number of virions (MV plus EV) for each replicate. Data shown represent average percent EV released calculated from each individual replicate set. (C) GARP KO cell lines were infected with MPXV WA. After 72 h, cells were fixed and stained with MPXV reactive anti-VACV antibody followed by HRP-conjugated secondary antibody. The monolayer was developed using o-dianisidine solution. Data are shown as means (n = 3), and error bars represent standard deviations. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 4

GARP complex genes are important for VACV EV production. GARP complex KO cell lines (VPS51KO, VPS52KO, VPS53KO, and VPS54KO) were infected with VACV strain IHDJ at an MOI of 1 in the absence and presence of 2 μM ST-246. MV and EV production was measured 24 hpi in each cell line. The percentage of EV released was determined by calculating the proportion of EV represented in the total number of virions (MV plus EV). All MV and EV values were averaged first, and then percent EV released was calculated from the averages. (C) Immunostaining of VACV IHDJ-infected KO cells. After 72 h, cells were fixed and stained with anti-VACV antibody followed by HRP-conjugated secondary antibody. Data are shown as means (n = 6), and error bars represent standard deviations. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 5

Complementation of GARP complex in KO cells restores viral production. (A) VPS52KO cells were transfected with pLenti-VPS52-FLAG plasmid (pVPS52) or control plasmid (CTRL). Cells were then infected with VACV strain IHDJ in the presence or absence of ST-246. MV and EV production was measured 24 hpi. The percentage of EV released was determined by calculating the proportion of EV represented in the total number of virions (MV plus EV). Data are shown as means (n = 7), and error bars represent standard deviations. (B) VPS52KO cells were transfected with a FLAG-tagged VPS52-expressing plasmid (pVPS52) or a non-VPS52-expressing plasmid (CTRL). After 24 h, cells were harvested and processed for Western blot analysis using anti-FLAG antibody followed by IRDye secondary antibodies and were visualized using direct infrared fluorescence. GAPDH was used as a control.

FIG 6

(A) Stable VPS54KO cell lines expressing empty vector or VPS54 were infected with VACV strain IHDJ in the presence or absence of 2 μM ST-246. MV and EV production was measured 24 hpi. The percentage of EV released was determined by calculating the proportion of EV represented in the total number of virions (MV plus EV). Data are shown as means (n = 3), and error bars represent standard deviations. (B) VPS54KO-empty and VPS54KO-VPS54 (V5-tagged) cells were harvested and processed for Western blot analysis using anti-V5 antibody, followed by HRP-conjugated secondary antibody. The blot was developed using enhanced chemiluminescent substrate. (C) Stable VPS54KO cell lines expressing empty vector or VPS54 were infected with VACV strain IHDJ. MV and EV production was measured at 0, 24, 48, 72, and 96 hpi. Data are shown as means (n = 3), and error bars represent standard deviations. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 7

Actin tail deficiency in VACV-infected VPS KO cells. Cell lines were infected with VACV WRA4-YFP virus (green) and stained with Alexa Fluor 647 phalloidin (red, to visualize F-actin) and DAPI (blue, to visualize DNA) after 24 h. Representative images of infection in VPS WT (upper left), VPS WT in the presence of 2 μM ST-246 (upper right), VPS52KO (bottom left), and VPS54KO (bottom right) cells are shown. White arrowheads point to a representative actin tail with loaded virus. Original magnification: ×40.

FIG 8

Transmission EM of VACV-infected VPS KO cells. (A) Representative images of VPS WT cells, VPS WT cells in the presence of 2 μM ST-246, VPS52KO cells, and VPS54KO cells infected with VACV IHDJ strain. An area representing the selected VPS WT is shown at higher magnification. Bar, 500 nm. The representative virion forms are labeled as follows: C, crescents; IV, immature virus; MV, mature virus; EV, extracellular virus; Nu, nucleus. (B) Quantification of percent WV in infected KO cells. A total of 20 to 25 electron microscopic images were examined for quantities of MV, WV, and EV. Comparisons of numbers of wrapped and extracellular virus to the total number of virions determined the percentage of WV present under each set of conditions.