Morgan Angelica L, Vu Michelle N, Zhou Yiyang, Lokugamage Kumari G, Meyers William M, Alvarado R Elias, Ahearn Yani, Estes Leah K, Plante Jessica A, Johnson Bryan A, Suthar Mehul S, Walker David H, Plante Ken S, Menachery Vineet D
Experimental Pathology Graduate Program, University of Texas Medical Branch, Galveston, Texas, USA.
Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
J Virol. 2025 Jun 10:e0046725. doi: 10.1128/jvi.00467-25.
The SARS-CoV-2 spike, key to viral entry, has two features that differentiate it from other sarbecoviruses: the presence of a furin cleavage site (FCS; PRRAR sequence) and an extended S1/S2 loop characterized by an upstream QTQTN amino acid motif. Our prior works show that shortening the S1/S2 loop by deleting either the FCS (ΔPRRA) or an upstream sequence (ΔQTQTN) ablates spike processing, alters host protease usage, and attenuates infection and . With the importance of the loop length established, we evaluated the impact of disrupting the FCS while preserving the S1/S2 loop length. Using reverse genetics, we generated a SARS-CoV-2 mutant that disrupts the FCS (PQQAR) but maintains its extended S1/S2 loop. The SARS-CoV-2 PQQAR mutant has reduced replication, decreased spike processing, and attenuated disease compared to wild-type SARS-CoV-2. These data, similar to those from the FCS deletion mutant, indicate that loss of the furin cleavage site attenuates SARS-CoV-2 pathogenesis. Importantly, we subsequently found that the PQQAR mutant can be transmitted in the direct contact hamster model despite lacking an intact FCS. However, competition transmission showed that the mutant was attenuated compared to WT SARS-CoV-2. Together, the data suggest that the FCS is required for SARS-CoV-2 pathogenesis but is not strictly required for viral transmission.
The presence of the furin cleavage site (FCS) within the spike protein of SARS-CoV-2 distinguishes it from other sarbecoviruses found in nature. While prior works have deleted the FCS, these mutant viruses also shortened the S1/S2 loop, which is known to be important for pathogenesis. This study defines the importance of the FCS in the context of the extended SARS-CoV-2 S1/S2 loop. The study finds that the FCS disruption mutant is attenuated and . Disruption of the FCS reduces spike processing and changes the usage of the host protease TMPRSS2. Importantly, while not strictly required, the FCS plays a role in SARS-CoV-2 transmission efficiency. Overall, the manuscript demonstrates the importance of the furin cleavage site for SARS-CoV-2 infection, pathogenesis, and transmission.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白是病毒进入细胞的关键，它有两个特征使其与其他沙贝病毒属病毒不同：存在弗林蛋白酶切割位点（FCS；PRRAR序列）以及以QTQTN上游氨基酸基序为特征的延长S1/S2环区。我们之前的研究表明，通过删除FCS（ΔPRRA）或上游序列（ΔQTQTN）来缩短S1/S2环区，会消除刺突蛋白的加工过程，改变宿主蛋白酶的使用情况，并减弱感染能力[参考文献1,2]。在确定了环区长度很重要之后，我们评估了在保留S1/S2环区长度时破坏FCS的影响。利用反向遗传学，我们构建了一种破坏FCS（PQQAR）但保留其延长S1/S2环区的SARS-CoV-2突变体。与野生型SARS-CoV-2相比，SARS-CoV-2 PQQAR突变体的复制能力降低，刺突蛋白加工减少，致病力减弱。这些数据与FCS缺失突变体的数据相似，表明弗林蛋白酶切割位点的缺失会减弱SARS-CoV-2的致病性。重要的是，我们随后发现，尽管PQQAR突变体缺乏完整的FCS，但它仍能在直接接触仓鼠模型中传播。然而，竞争传播实验表明，与野生型SARS-CoV-2相比，该突变体的传播能力减弱了。总之，这些数据表明FCS是SARS-CoV-2致病所必需的，但不是病毒传播所严格必需的。
SARS-CoV-2刺突蛋白中弗林蛋白酶切割位点（FCS）的存在使其与自然界中发现的其他沙贝病毒属病毒区分开来。虽然之前的研究删除了FCS，但这些突变病毒也缩短了S1/S2环区长度，而该环区已知对致病性很重要。本研究确定了在延长SARS-CoV-2 S1/S2环区背景下FCS的重要性。该研究发现FCS破坏突变体的致病力减弱[参考文献1,2]。FCS的破坏会减少刺突蛋白的加工，并改变宿主蛋白酶TMPRSS2的使用情况。重要的是虽然不是严格必需的，但FCS在SARS-CoV-2的传播效率中发挥作用。总体而言，该论文证明了弗林蛋白酶切割位点对SARS-CoV-2感染、致病性和传播的重要性。
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