Centre International de Recherche en Infectiologie, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Université Lyon, Lyon, France.
Plateforme IBiSA de Microscopie Electronique, Université de Tours et CHU de Tours, Tours, France.
J Virol. 2019 Jan 4;93(2). doi: 10.1128/JVI.01716-18. Print 2019 Jan 15.
The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3. IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.
干扰素诱导的跨膜蛋白（IFITMs）是一组高度相关的抗病毒因子，它们在两个步骤中影响多种病毒：在靶细胞中，通过将进入的病毒隔离在内体中，以及在产生细胞中，通过导致包装 IFITMs 的病毒颗粒的产生，从而表现出降低的感染性。虽然大多数研究都集中在前一种情况，但关于 IFITM 对病毒颗粒感染性的负印迹的调节及其与靶细胞保护的关系知之甚少。我们使用一组针对 HIV-1 的 IFITM3 突变体来探索这些问题以及与 IFITM3 生物学相关的其他问题，特别是病毒颗粒包装、稳定性、与 CD63/多泡体（MVBs）的关系、胆固醇水平的调节以及病毒颗粒负印迹与靶细胞保护之间的关系。我们获得的结果排除了胆固醇的作用，并表明 CD63 积累与抗病毒行为没有直接关系。我们已经定义了调节 IFITM3 两种抗病毒特性的区域以及调节蛋白质稳定性的新区域，从而影响其包装到病毒颗粒中的程度。然而，我们获得的结果表明，即使在 IFITM 易感病毒的情况下，IFITM3 包装也不足以进行负印迹。最后，虽然大多数突变同时影响靶细胞保护和负印迹，但 C 末端结构域（CTD）中的一个区域表现出不同的行为，这可能突出了该结构域在 IFITM3 的两种抗病毒活性中可能发挥的调节作用。IFITM 蛋白与进入的病毒颗粒在内体中的隔离（靶细胞保护）以及产生包含 IFITM 并表现出降低感染性的病毒颗粒（病毒颗粒感染性的负印迹）有关。后者是如何调节的，以及这两种抗病毒特性是否相关，仍然未知。通过研究针对 HIV-1 的大量 IFITM3 突变体的行为，我们确定 IFITM3 突变体基本上与它们在细胞内的表达水平成比例地包装到病毒颗粒中。然而，即使在 IFITM 易感病毒的情况下，IFITM3 包装对于抗病毒作用也不充分。大多数突变被发现同时影响 IFITM3 的两种抗病毒特性，但一个 CTD 突变体表现出不同的行为，这可能突出了该结构域的新调节作用。这些发现因此提高了我们对这类广谱抗病毒限制因子如何作用的理解。
