Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.
Monkeypox virus(MPXV) is a human pathogen that is a member of theOrthopoxvirusgenus, which includesVaccinia virusandVariola 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 thetrans-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.IMPORTANCEHuman monkeypox is an emerging zoonotic infectious disease caused byMonkeypox 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.
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Susan
S
Realegeno
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
Andreas S
AS
Puschnik
Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Amrita
A
Kumar
Infectious Disease Pathology Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Cynthia
C
Goldsmith
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Jillybeth
J
Burgado
Immunology and Pathogenesis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Suryaprakash
S
Sambhara
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Victoria A
VA
Olson
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Darin
D
Carroll
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Inger
I
Damon
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Tetsuya
T
Hirata
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Taroh
T
Kinoshita
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA carette@stanford.edu spanayampalli@cdc.gov.
Jan E
JE
Carette
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA carette@stanford.edu spanayampalli@cdc.gov.
Panayampalli Subbian
PS
Satheshkumar
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001692:Biological Transport; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D015894:Genome, Human; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000235:genetics; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D006238:Haploidy; D054884:Host-Pathogen Interactions; D006801:Humans; D054833:Isoindoles / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D045908:Mpox, Monkeypox / Q000821:virology; D008993:Monkeypox virus / Q000502:physiology*; D016254:Mutagenesis, Insertional; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D019562:Viral Load; D014779:Virus Replication; D010797:Phthalimides
D000199:Actins; D001549:Benzamides; D006025:Glycosaminoglycans; D017261:Glycosylphosphatidylinositols; D054833:Isoindoles; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C087215:LRRC32 protein, human; C503307:VPS53 protein, human; C505045:tecovirimat; D010797:Phthalimides
GARP complex; HAP1 screen; poxviruses; retrograde transport