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

Abstract

(MPXV) is a human pathogen that is a member of the genus, which includes and (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-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. Human monkeypox is an emerging zoonotic infectious disease caused by (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.

TMEM41B is a pan-flavivirus host factor

“…We hypothesized that this alteration in surface protein glycosylation would decrease the enrichment and overabundance of hits related to HS protein glycosylation and nonspecific virus binding. Nevertheless, consistent with a wide range of virus-selected genome-wide loss of function screens (Hoffmann et al, 2017;Jae et al, 2014;Realegeno et al, 2017;Riblett et al, 2016), we identified host factors involved in heparan sulfate biosynthesis.…”

Report of Monkeypox cases in 2018 in the United Kingdom

“…The vaccine against smallpox in humans has been shown to be 85% effective against monkeypox (21) but issues with vaccine safety and relatively small numbers of cases have not justified vaccination. Further, monkeypox is occurring in areas which struggle to maintain adequate vaccine coverage levels for routine vaccinations against measles and polio, which remain a higher priority (41). The global increase in monkeypox should be a cause of concern.…”

Discovery of Retro-1 Analogs Exhibiting Enhanced Anti-vaccinia Virus Activity

“…While several viruses (Simian virus 40, papillomavirus, and influenza) rely on retrograde transport for entry into a cell, OPXVs require retrograde transport for EV membrane wrapping, and consequently viral egress (Spooner et al, 2006;Harrison et al, 2016;Sivan et al, 2016). We and others have previously demonstrated that impeding the retrograde pathway by targeting the Golgi-associated retrograde protein complex can greatly reduce EV yield with minimal impact on MV formation in VACV and MPXV (Harrison et al, 2016;Realegeno et al, 2017). Treatment with small molecule inhibitors of the retrograde pathway such as Retro-1 and Retro-2 significantly decreased EV formation in vitro by preventing the trafficking of the viral protein F13, which is required for membrane wrapping (Harrison et al, 2016;Sivan et al, 2016).…”