Lin David L, Cherepanova Natalia A, Bozzacco Leonia, MacDonald Margaret R, Gilmore Reid, Tai Andrew W
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
mBio. 2017 Jul 18;8(4):e00939-17. doi: 10.1128/mBio.00939-17.
Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV. The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.
登革病毒（DENV）是全球最常见的虫媒病毒感染，每年估计感染3.9亿人。我们采用全基因组成簇规律间隔短回文重复序列（CRISPR）筛选来鉴定DENV增殖所需的宿主依赖因子，并确定寡糖基转移酶（OST）复合体是DENV感染的必需宿主因子。哺乳动物细胞表达两种含有STT3A或STT3B的OST。我们发现，OST作为寡糖基转移酶的典型催化功能对DENV感染并非必需，因为表达无催化活性的STT3A或STT3B的细胞能够支持DENV增殖。然而，与STT3B相关的OST亚基MAGT1对DENV增殖也是必需的。MAGT1的表达需要STT3B，且无催化活性的STT3B也能挽救MAGT1的表达，这支持了在DENV感染情况下STT3B用于稳定MAGT1的假说。我们发现MAGT1的氧化还原酶CXXC活性位点基序对DENV增殖是必需的，因为表达AXXA MAGT1突变体的细胞无法支持DENV感染。有趣的是，表达MAGT1的单半胱氨酸CXXA或AXXC突变体的细胞能够支持DENV增殖。利用工程过氧化物酶APEX2，我们证明了在DENV感染期间MAGT1与NS1或NS4B紧密相邻。这些结果表明，含STT3B的OST的氧化还原酶活性对DENV感染是必需的，这可能会指导针对DENV的抗病毒药物的开发。宿主寡糖基转移酶（OST）复合体已被确定为登革病毒（DENV）复制的必需宿主因子；然而，它们在DENV感染期间的功能尚不清楚。先前的一项研究表明，典型的OST活性对DENV复制是可有可无的，这表明OST复合体作为DENV复制的支架。然而，我们的研究表明，OST复合体在DENV感染期间的一个功能是通过OST亚基MAGT1提供氧化还原酶活性。我们还表明，在病毒感染期间MAGT1与DENV NS1和NS4B相关联，这表明这些非结构蛋白可能是MAGT1氧化还原酶活性的靶点。这些结果为DENV感染的细胞生物学提供了见解，这可能会指导针对DENV的抗病毒药物的开发。
