Mannsverk Steinar, Villamil Giraldo Ana M, Kasson Peter M
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA.
J Virol. 2025 Jul 24:e0110525. doi: 10.1128/jvi.01105-25.
Many enveloped viruses enter cells via fusion with the endosomal membrane, raising the question whether entry through the endosomal route confers a fitness advantage over fusion directly at the plasma membrane. We found that influenza A virus fusion at the plasma membrane of A549 cells resulted in a 4.5-fold reduction in productive cell infection, compared to infection through the physiological endosomal route. We hypothesized that this was partially explained by restrictive and permissive membrane factors at the plasma and endosomal membrane, respectively. To test this, we developed a single-viral content mixing assay with plasma membrane vesicles (PMVs), where a fluorescent content marker was loaded into purified PMVs at a quenched concentration, through freeze-thawing. We show that influenza fusion with the plasma membrane is sixfold less efficient than with liposomes containing the endosome-enriched phospholipid BMP. Incorporating BMP into the PMVs with pre-loaded methyl-α-cyclodextrin (MαCD) did not rescue full fusion, suggesting that the lack of BMP in the plasma membrane is not sufficient to explain decreased fusion with the plasma membrane. Depletion of cholesterol from PMVs enhanced lipid mixing rates and reduced membrane order. Lastly, modest depletion of cholesterol from A549 lung epithelial cells rescued influenza infection through fusion at the plasma membrane by 2.4-fold. We propose that for the endosome-adapted influenza virus, fusion directly at the plasma membrane is restricted by the liquid-ordered-like nature of the plasma membrane, raising the question whether the plasma membrane is broadly less permissive to endosome-adapted viruses.IMPORTANCEInfluenza virus normally enters cells via endosomes, yet it has been known for decades that acidic conditions can force it to enter via the plasma membrane. Here, we show that the plasma membrane route yields 4.5-fold worse infection of cells, and surprisingly, most of this effect can be attributed to the plasma membrane being less permissive to viral membrane fusion. This difference between plasma membrane and endosomes cannot be simply abolished by adding a key endosomal lipid that can boost fusion in synthetic membranes. The outer surface of the plasma membrane is known to be highly ordered, and disrupting this order does increase fusion as well as boost the ability of influenza virus to infect cells. We, therefore, conclude that the complex and ordered composition of cell membranes helps control when and where influenza virus can enter and infect.
许多包膜病毒通过与内体膜融合进入细胞，这就引发了一个问题：通过内体途径进入细胞是否比直接在质膜处融合具有适应性优势。我们发现，甲型流感病毒在A549细胞质膜处融合导致有效细胞感染率降低了4.5倍，而通过生理性内体途径感染的情况则不然。我们推测，这部分是由于质膜和内体膜上分别存在限制性和允许性膜因子所致。为了验证这一点，我们开发了一种针对质膜囊泡（PMV）的单病毒内容物混合检测方法，其中通过冻融将荧光内容物标记物以淬灭浓度加载到纯化的PMV中。我们发现，流感病毒与质膜的融合效率比与含有内体富集磷脂BMP的脂质体的融合效率低六倍。用预加载的甲基-α-环糊精（MαCD）将BMP掺入PMV中并不能挽救完全融合，这表明质膜中缺乏BMP不足以解释与质膜融合减少的现象。从PMV中去除胆固醇可提高脂质混合速率并降低膜有序性。最后，从A549肺上皮细胞中适度去除胆固醇可使流感病毒通过在质膜处融合进行感染的能力提高2.4倍。我们提出，对于适应内体的流感病毒而言，直接在质膜处融合受到质膜类似液态有序性质的限制，这就引发了一个问题：质膜对适应内体的病毒是否普遍更不具有允许性。
重要性
流感病毒通常通过内体进入细胞，但几十年来人们已知酸性条件可迫使它通过质膜进入。在这里，我们表明质膜途径导致细胞感染率降低4.5倍，令人惊讶的是，这种影响大部分可归因于质膜对病毒膜融合的允许性较低。质膜和内体之间的这种差异不能通过添加一种能促进合成膜中融合的关键内体脂质而简单消除。已知质膜外表面高度有序，破坏这种有序性确实会增加融合以及增强流感病毒感染细胞的能力。因此，我们得出结论，细胞膜复杂且有序的组成有助于控制流感病毒何时何地能够进入并感染细胞。
