Mechanisms of Virus Entry into Cells and Antiviral Barriers Limiting Entry

自2017年2月开始该项目的工作以来，我们已经产生了大量新的IFITM 3突变体来帮助我们的研究。此外，我们已经开发了使用MLV为基础的载体系统，并采用了假型化程序来研究各种逆转录病毒糖蛋白如何影响病毒对IFITM介导的抗病毒活性的易感性。迄今为止，我们的数据表明IFITM 3在抑制逆转录病毒颗粒感染性的能力方面表现出很大的广度。这些发现表明，这些抗病毒蛋白质可能会对不同物种中具有医学重要性的多种哺乳动物逆转录病毒造成严重障碍。具体地，IFITM 3在病毒产生细胞中的存在导致包膜糖蛋白水平降低，这导致病毒颗粒显示出非常少的与细胞靶标的附着和进行融合所需的糖蛋白。这项工作的第一部分已经出版（Ahi et al.，mBio 11：e03088-19，2020）。我们证明了IFITM 3在异位或内源性表达时负调节病毒糖蛋白，因为RNAi和CRISPR/Cas9介导的IFITM 3在人细胞和鼠细胞中的基因敲除导致病毒糖蛋白的半衰期更长。此外，IFITM 3通过干扰病毒糖蛋白的顺行运输并将其重定向至溶酶体进行降解来抑制病毒糖蛋白。我们发现，病毒糖蛋白的抑制作用是广泛的（影响HIV-1 Env、MLV Env和VSV-糖蛋白），而埃博拉病毒糖蛋白是耐药的。此外，许多细胞糖蛋白不受IFITM 3的影响。这一发现揭示了IFITM 3利用一般机制广泛抑制病毒融合和传播所需的病毒糖蛋白的产生。我们还鉴定了一些IFITM 3突变体，它们失去了这种抗病毒功能。我们目前正在使用结构同源性来预测这些突变位点所属的功能基序，这将有助于我们理解单个突变如何以及为什么会破坏活性。最近，我们在细胞内环（G95 L）中发现了一个单残基突变，该突变消除了这种抗病毒功能。我们发现G95形成蛋白质寡聚化所需的二甘氨酸基序的一部分。我们的发现表明IFITM 3寡聚体负责抑制其两种抗病毒活性（抑制病毒进入幼稚细胞和抑制病毒产生细胞中的病毒体感染性）（Rahman等人，提交）。总之，这些努力将为IFITM蛋白的功能以及它们所属的扩展的CD 225蛋白家族提供广泛的见解，并将为开发新的抗病毒疗法提供杠杆作用。

Since work on this project begun in February 2017, we have generated a large panel of novel IFITM3 mutants to aid our study. Furthermore, we have developed systems using MLV-based vectors and adapted a pseudotyping procedure to study how various retroviral glycoproteins affect viral susceptibility to IFITM-mediated antiviral activities. Our data thus far suggest that IFITM3 exhibits great breadth with regard to its ability to inhibit retroviral particle infectivity. These findings suggest that these antiviral proteins may impose serious barriers to multiple mammalian retroviruses of medical importance in different species. Specifically, the presence of IFITM3 in virus-producing cells leads to decreased levels of envelope glycoprotein, which results in virus particles displaying very little glycoprotein needed to attach and perform fusion with cell targets. The first installment of this work has been published (Ahi et al., mBio 11: e03088-19, 2020). We demonstrated that IFITM3 negatively regulates viral glycoproteins when expressed ectopically or endogenously, as RNAi and CRISPR/Cas9-mediated gene knockout of IFITM3 in human cells and in murine cells results in longer half-lives of viral glycoproteins. Furthermore, IFITM3 inhibits viral glycoproteins by interfering with their anterograde trafficking and redirecting them to lysosomes for degradation. We found that inhibition of viral glycoproteins is broad (affecting HIV-1 Env, MLV Env, and VSV-glycoprotein), while Ebolavirus glycoprotein is resistant. Furthermore, a number of cellular glycoproteins are unaffected by IFITM3. This finding reveals that IFITM3 utilizes a general mechanism to broadly inhibit the production of viral glycoproteins needed for virus fusion and spread. We have also identified a number of IFITM3 mutants that have lost this antiviral function. We are currently using structural homology to predict functional motifs to which these mutated sites belong, which will facilitate our understanding of how and why individual mutations disrupt activity. Most recently, we identified a single residue mutation in the intracellular loop (G95L) that abrogates this antiviral function. We found that G95 forms part of a di-glycine motif needed for protein oligomerization. Our findings indicate that IFITM3 oligomers are responsible for the inhibition of both of its antiviral activities (inhibition of virus entry into naive cells and inhibition of virion infectivity in virus-producing cells) (Rahman et al., submitted). Together, these efforts will provide extensive insight into the function of IFITM proteins as well as the extended CD225 protein family to which they belong and will provide leverage for the development of new antiviral therapies.