The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses.

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Effector proteins injected into the host cell by the C. burnetii type IVB secretion system (T4BSS) are required for the inhibition of host cell apoptosis. AnkG is one of these anti-apoptotic effector proteins. The inhibitory effect of AnkG requires its nuclear localization, which depends on p32-dependent intracellular trafficking and importin-α1-mediated nuclear entry of AnkG. Here, we compared the sequences of ankG from 37 C. burnetii isolates and classified them in three groups based on the predicted protein size. The comparison of the three different groups allowed us to identify the first 28 amino acids as essential and sufficient for the anti-apoptotic activity of AnkG. Importantly, only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Finally, using the Galleria mellonella infection model, we observed that AnkG from the first group has the ability to attenuate pathology during in vivo infection, as it allows survival of the larvae despite bacterial replication.