文章分类：热点透视 发布时间:2021-08-22 ACTV 阅读( )
Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with severe acute respiratory syndrome (SARS)-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two Middle East respiratory syndrome (MERS)-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate the infection of human gut cells but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that the addition of exogenous trypsin also rescues HKU5-CoV, a second bat group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate the emergence potential of bat CoVs and offers a means to recover previously unrecoverable zoonotic CoV strains.
Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus
MERS-CoV, PDF2180, coronavirus, emergence, spike, zoonotic
Despite their spontaneous emergence, several research approaches to rapidly respond and even predict outbreak strains already exist. During the MERS-CoV outbreak, our group and others were able to leverage reagents generated against related group 2C coronaviruses, namely, HKU4- and HKU5-CoV (6, 7). These reagents, created independent of viable virus replication, provided valuable insights and models for testing serologic responses during the early stages of the MERS-CoV outbreak. Similarly, reverse genetics systems permitted the exploration of zoonotic coronaviruses (8); using the known SARS spike/ACE2 receptor interaction, chimeric viruses containing the backbones of bat CoVs were generated to evaluate the efficacy of both vaccines and therapeutics (9–12). The inverse approach placed the zoonotic spike proteins in the context of the epidemic SARS-CoV backbone (13, 14). These studies provided insight into potential threats circulating in bats as well as the efficacy of current therapeutic treatments (15). While far from comprehensive, the results indicated that these approaches, reagents, and predictions may prove useful in preparations for future CoV outbreaks.
In this study, we extend the examination of zoonotic viruses to a novel MERS-like CoV strain isolated from a Ugandan bat, namely, PDF-2180 CoV (MERS-Uganda). Our initial attempt to cultivate a chimeric MERS-CoV containing the Ugandan MERS-like spike produced viral subgenomic transcripts but failed to result in infectious virus after electroporation (16). However, in the current study, we demonstrate that exogenous trypsin treatment produced high-titer virus capable of plaque formation and continued replication. These results are consistent with the recovery of enteric CoVs like porcine epidemic diarrhea virus (17) but have not previously been described as a major barrier for bat-derived CoVs. The chimeric Ugandan MERS-like spike virus could replicate efficiently in both Vero and Huh7 cells in the context of trypsin-containing media but failed to produce infection of either continuous or primary human respiratory cell cultures. Importantly, the MERS-Uganda chimeric virus successfully infected cells of the human digestive tract, potentially identifying another route for cross-species transmission and emergence. Notably, blockade of human DPP4, the receptor for MERS-CoV, had no significant impact on replication of the chimeric MERS-Uganda virus, suggesting the use of an alternative receptor. Similarly, the addition of trypsin also rescued replication of full-length HKU5-CoV, a related group 2C bat CoV, and showed no replication defect during DPP4 blockade. Together, the results indicate that proteolytic activation of the spike protein is a potent constraint to infection for zoonotic CoVs and expand the correlates for CoV emergence beyond receptor binding alone. (This article was submitted to an online preprint archive .)
Exploring viral genomic RNA, trypsin in the culture media permitted robust infection with HKU5-CoV that increased over time and was absent in cells not treated with trypsin (Fig. 6A). Similarly, trypsin in the media also permitted the accumulation and proteolytic cleavage of the HKU5 spike protein in a dose- and time-dependent manner (Fig. 6B). Importantly, the addition of the anti-DPP4 antibody had no impact on HKU5-CoV infection, suggesting the use of a different receptor than that used by wild-type MERS-CoV, similar to the findings with the MERS-Uganda spike (Fig. 6C). Together, these results demonstrate that protease cleavage is also the primary barrier to infection of Vero cells with HKU5-CoV. Examining further, we compared the predicted cleavage at S1/S2 border, S2’, and the endosomal cysteine protease site across MERS, PDF2180, and HKU5 spikes (Fig. 6D) (26). For the S1/S2 site, MERS, Uganda, and HKU5 maintain the RXXR cleavage motif, although the different interior amino acids may alter efficiency. For the S2’ sequence, MERS and HKU5 also retain the RXXR motif; however, the Uganda spike lacks the first arginine (SNAR), potentially impacting cleavage. Finally, all three spikes maintain an aromatic residue at position two in the endosomal cysteine protease (ECP) site (27). However, the HKU5 spike maintains N at position 1 which is similar to a MERS mutant previously shown to inactivate cathepsin L activation (28). Together, the results suggest that potential sequence changes in the protease cleavage sites may contribute to the trypsin dependency of MERS-Uganda and HKU5 spike-mediated infections.
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