Discovery of orally bioavailable SARS-CoV-2 papain-like protease inhibitor as a potential treatment for COVID-19 (preprint)

The RNA-dependent RNA polymerase (RdRp), 3C-like protease (3CLpro), and papain-like protease (PLpro) are pivotal components in the viral life cycle of SARS-CoV-2, presenting as promising therapeutic targets. Currently, all FDA-approved antiviral drugs against SARS-CoV-2 are RdRp or 3CLpro inhibitors. However, the mutations causing drug resistance have been observed in RdRp and 3CLpro from SARS-CoV-2, which makes it necessary to develop antivirals with novel mechanisms. Through the application of a structure-based drug design (SBDD) approach, we discovered a series of novel potent non-covalent PLpro inhibitors with remarkable in vitro potency and in vivo PK properties. The co-crystal structures of PLpro with leads revealed that the residues E164 and Q269 around the S2 site are critical for improving the inhibitor\'s potency. The lead compound GZNL-P36 not only inhibited SARS-CoV-2 and its variants at the cellular level with EC50 ranging from 58.2 nM to 306.2 nM, but also inhibited HCoV-NL63 and HCoV-229E with EC50 of 81.6 nM and 2.66 M, respectively. Oral administration of the compound resulted in significantly improved survival and notable reductions in lung viral loads and lesions in SARS-CoV-2 infection mouse model, consistent with RNA-seq data analysis. Our results indicate that PLpro inhibitor is a promising SARS-CoV-2 therapy.

Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses (preprint)

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens (preprint)

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

The adenosine analogue prodrug ATV006 is orally bioavailable and has potent preclinical efficacy against SARS-CoV-2 and its variants (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5-hydroxyl -isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.

NSUN2-mediated m5C methylation of IRF3 mRNA negatively regulates type I interferon responses (preprint)

5-Methylcytosine (m 5 C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m 5 C methyltransferase, can negatively regulate type I interferon responses during viral infection. NSUN2 specifically mediates m 5 C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-β production. Knockout or knockdown of NSUN2 could enhance type I interferon responses and downstream ISG expression after viral infection in vitro . And in vivo , the antiviral innate responses is more dramatically enhanced in Nsun2 +/− mice than in Nsun2 +/+ mice. Four highly m 5 C methylated cytosines in IRF3 mRNA were identified, and their mutation could enhance the cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), Zika virus (ZIKV), or especially SARS-CoV-2 resulted in a reduction in endogenous levels of NSUN2. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease after viral infection to boost antiviral responses for the effective elimination of viruses. Our results suggest a paradigm of innate antiviral immune responses ingeniously involving NSUN2-mediated m 5 C modification.

Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication (preprint)

Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral proteins for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. In summary, our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

Co-Infecting Pathogens Can Contribute to Inflammatory Responses and Severe Symptoms in COVID-19 (preprint)

Background: The current pandemic of COVID-19 is posing a major challenge to public health on a global scale. While it is generally believed severe COVID-19 results from over-expression of inflammatory mediators (i.e. a “cytokine storm”), it is still unclear whether and how co-infecting pathogens contribute to disease pathogenesis. To address this, we followed the entire course of disease in severe COVID-19 cases to reveal the presence and abundance of all potential pathogens present - the total “infectome” - and how they interact with the host immune system in the context of severe COVID-19 disease.Methods: We considered one severe and three critical cases of COVID-19, as well as a set of healthy controls, with longitudinal samples (throat swab, whole blood and serum) taken in each case. Total RNA sequencing (meta-transcriptomics) was performed to simultaneously reveal pathogen diversity and abundance, as well as host immune responses, within each sample. A Bio-Plex method was used to measure serum cytokine and chemokine levels.Findings: Eight pathogens were identified in these COVID-19 patients - Aspergillus fumigatus, Mycoplasma orale, Myroides odorantus, Acinetobacter baumannii, Candida tropicalis, herpes simplex virus and human cytomegalovirus - that appeared at different stages of disease course. Notably, the dynamics of inflammatory mediators in the serum as well as respiratory tract were better associated with the dynamics of the infectome as a whole rather than SARS-CoV-2 alone. Correlation analysis revealed that pulmonary injury was directly associated with cytokine levels, which in turn was associated with the proliferation of SARS-CoV-2 and the co-infecting pathogens.Interpretation: The cytokine storm that resulted in aggravated acute lung injury and death involved the highly complex and dynamic entire infectome of each patient, of which SARS-CoV-2 was a component. These results call for a precision-medicine approach to investigating both the infection and the host response on a daily basis as a standard means of infectious disease characterization.Funding: Guangzhou Institute of Respiratory Health Open Project (Funds provided by China Evergrande Group) - Project No. (2020GIRHHMS01), Guangdong Province “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project (2019ZT08Y464), Macao Science and Technology Development Fund (0042/2020/A), Science research project of the Guangdong Province (2019B030316028), Special Project for Scientific and Technological Development and Emergency Response in COVID-19 Prevention and Control of Guangdong Province (2020A111129028), Special Project for Research and Promotion of Prevention and Control Techniques of COVID-19 and Emergency Response in Dongguan City (202071715001114), Jack Ma Foundation (2020-CMKYGG-02), Guangzhou Medical University High-level University Clinical Research and Cultivation Program ([2017] 159 and 160) and ARC Australian Laureate Fellowship (FL170100022).Declaration of Interests: We declare no competing interests.Ethics Approval Statement: The ethics committee of the FAHGMU (Ethics No. 2020-85) and Dongguan’s People’s Hospital (KYKT2020-005-A1) approved the sampling procedure and the use of patient samples for this study. Informed consent was obtained from each patient.

SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway (preprint)

Cytokine storm resulting from a heightened inflammatory response is a prominent feature of severe COVID-19 disease. This inflammatory response results from assembly/activation of a cell-intrinsic defense platform known as the inflammasome. We report that the SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. ORF3a triggers IL-1 beta expression via NFkB, thus priming the inflammasome while also activating it via ASC-dependent and -independent modes. ORF3a-mediated inflammasome activation requires efflux of potassium ions and oligomerization between NEK7 and NLRP3. With the selective NLRP3 inhibitor MCC950 able to block ORF3a-mediated inflammasome activation and key ORF3a residues needed for virus release and inflammasome activation conserved in SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention.

Remdesivir Metabolite GS-441524 Efficiently Inhibits SARS-CoV-2 Infection in Mouse Model (preprint)

The outbreak of coronavirus disease 2019 (COVID-19) rapidly spreads across worldwide and becomes a global pandemic. Remdesivir is the only COVID-19 treatment approved by U.S. Food and Drug Administration (FDA); however, its effectiveness is still under questioning as raised by the results of a large WHO Solidarity Trial. Herein, we report that the parent nucleotide of remdesivir, GS-441524, potently inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero E6 and other cells. It exhibits good plasma distribution and longer half-life (t1/2=4.8h) in rat PK study. GS-441524 is highly efficacious against SARS-CoV-2 in AAV-hACE2 transduced mice and murine hepatitis virus (MHV) in mice, reducing the viral titers in CoV-attacked organs, without noticeable toxicity. Given that GS-441524 was the predominant metabolite of remdesivir in the plasma, the anti-COVID-19 effect of remdesivir may partly come from the effect of GS-441524. Our results also supported that GS-441524 as a promising and inexpensive drug candidate in the treatment of COVID-19 and future emerging CoVs diseases.