Realegeno Susan, Puschnik Andreas S, Kumar Amrita, Goldsmith Cynthia, Burgado Jillybeth, Sambhara Suryaprakash, Olson Victoria A, Carroll Darin, Damon Inger, Hirata Tetsuya, Kinoshita Taroh, Carette Jan E, Satheshkumar Panayampalli Subbian
Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
J Virol. 2017 May 12;91(11). doi: 10.1128/JVI.00011-17. Print 2017 Jun 1.
(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.
猴痘病毒（MPXV）是一种人类病原体，属于痘病毒属，该属还包括天花病毒（导致天花的病原体）。人类猴痘被认为是一种新出现的人畜共患传染病。为了确定MPXV感染所需的宿主因子，我们在人类单倍体细胞中进行了全基因组插入诱变筛选。该筛选揭示了几个候选基因，包括参与高尔基体运输、糖胺聚糖生物合成和糖基磷脂酰肌醇（GPI）锚定生物合成的基因。我们验证了一组液泡蛋白分选（VPS）基因在感染过程中的作用，即VPS51至VPS54（VPS51 - 54），它们组成了高尔基体相关逆行蛋白（GARP）复合体。GARP复合体是一种拴系复合体，参与内体向高尔基体的逆行运输。我们的数据表明，VPS52和VPS54对于成熟病毒粒子（MV）的产生不是必需的，但对于细胞外病毒（EV）的形成是必需的。为了进行比较，我们在实验中使用了一种已知的抗病毒化合物ST - 246，结果表明VPS52和VPS54基因敲除（KO）细胞中的EV滴度与ST - 246处理的野生型细胞所表现出的水平相当。共聚焦显微镜用于检查肌动蛋白尾的形成，这是病毒在细胞间传播的一种出芽机制，结果显示在VPS52基因敲除或VPS54基因敲除感染的细胞中不存在肌动蛋白尾。通过电子显微镜对这些细胞的进一步评估表明，与野生型对照相比，包裹病毒（WVs）的水平有所下降。总体而言，我们的数据证明了GARP复合体基因在EV形成所必需的MV双膜包裹中的作用，这表明宿主内体运输途径参与了正痘病毒感染。人类猴痘是由猴痘病毒（MPXV）引起的一种新出现的人畜共患传染病。在MPXV的两个进化分支中，刚果盆地毒株与严重疾病、死亡率增加以及相对于西非毒株而言更高的人际传播率相关。猴痘在非洲西部和中部地区流行，但在2003年因进口受感染动物而传入美国。由于缺乏常规的天花疫苗接种，导致未接触过疫苗的人群比例更高，MPXV和其他正痘病毒的威胁正在增加。在这项研究中，我们已经鉴定并验证了MPXV感染所需的候选基因，具体而言，是那些与高尔基体相关逆行蛋白（GARP）复合体相关的基因。确定感染所需的宿主靶点，如GARP复合体或逆行途径，可防止细胞外病毒形成，这可为抗病毒治疗提供潜在靶点。
