Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.

IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.

干扰素诱导的跨膜蛋白（IFITM）是抑制许多包膜RNA病毒感染进入的限制因子。但是，我们之前证明了人类IFITM2和IFITM3是促进人类冠状病毒（HCoV）OC43进入的必不可少的宿主因子。在继续努力解释潜在的IFITM差异调节HCoV进入的分子机制的过程中，我们研究了对IFITM蛋白翻译后修饰，细胞内运输和寡聚化重要的结构基序在调节5个HCoV进入中的作用。我们发现，IFITM1或IFITM3中的三个不同突变可转化宿主限制性因子，以增强由严重急性呼吸综合征冠状病毒（SARS-CoV）和/或中东呼吸综合征冠状病毒（MERS-CoV）的突突蛋白驱动的进入。首先，用丙氨酸或天冬氨酸替代IFITM3酪氨酸20以模拟未磷酸化或磷酸化的IFITM3降低了其抑制HCoV-NL63和-229E进入的活性，但增强了SARS-CoV和MERS-CoV的进入。其次，用丙氨酸或天冬氨酸替代IFITM3酪氨酸99降低了其抑制HCoV-NL63和SARS-CoV进入的活性，但促进了MERS-CoV的进入。第三，从IFITM1中删除羧基末端的12个氨基酸残基增强了MERS-CoV和HCoV-OC43的进入。

重要信息即使使用同一受体，IFITM蛋白对利用不同进入途径和膜融合机制的HCoV进入的不同作用也使HCoVs成为有价值的系统，可用于比较研究IFITM限制或促进病毒进入宿主的分子机制细胞。鉴定了将IFITM1或IFITM3从抑制剂转变为MERS-CoV或SARS-CoV穗蛋白介导的增强子的三个不同突变，揭示了决定IFITM蛋白生物学活性的关键结构基序或残基。因此，这些发现为进一步鉴定与IFITM蛋白质的结构基序相互作用的病毒和宿主因子铺平了道路，从而差异性调节HCoV的感染性进入。