Structural and biochemical mechanism for increased infectivity and immune evasion of Omicron BA.1 and BA.2 variants and their mouse origins (preprint)

The Omicron BA.2 variant has become a dominant infective strain worldwide. Receptor binding studies reveal that the BA.2 spike trimer have 11-fold and 2-fold higher potency to human ACE2 than the spike trimer from the wildtype and Omicron BA.1 strains. The structure of the BA.2 spike timer reveals that all three receptor-binding domains (RBD) in the spike trimer are in open conformation, ready for high affinity binding to human ACE2, providing the basis for the increased infectivity of the BA.2 strain. JMB2002, a therapeutic antibody that was shown to have efficient inhibition of Omicron BA.1, also shows potent neutralization activities against Omicron BA.2. In addition, both BA.1 and BA.2 spike trimers are able to bind to the mouse ACE2 with high potency. In contrast, the wildtype spike trimer binds well to cat ACE2 but not to mouse ACE2. The structures of both BA.1 and BA.2 spike trimer bound to mouse ACE2 reveal the basis for their high affinity interactions. Together, these results suggest a possible evolution pathway for Omicron BA.1 and BA.2 variants from human-cat-mouse-human circle, which could have important implications in establishing an effective strategy in combating viral infection.

Structural basis for repurpose and design of nucleoside drugs for treating COVID-19 (preprint)

SARS-CoV-2 has caused a global pandemic of COVID-19 that urgently needs an effective treatment. Nucleoside analog drugs including favipiravir have been repurposed for COVID-19 despite of unclear mechanism of their inhibition of the viral RNA polymerase (RdRp). Here we report the cryo-EM structures of the viral RdRp in complex with favipiravir and two other nucleoside inhibitor drugs ribavirin and penciclovir. Ribavirin and the ribosylated form of favipiravir share a similar ribose scaffold that is distinct from penciclovir. However, the structures reveal that all three inhibitors are covalently linked to the primer strand in a monophosphate form despite the different chemical scaffolds between favipiravir and penciclovir. Surprisingly, the base moieties of these inhibitors can form mismatched pairs with the template strand. Moreover, in view of the clinical disadvantages of remdesivir mainly associated with its prodrug form, we designed several orally-available remdesivir parent nucleoside derivatives, including VV16 that showed 5-fold more potent than remdesivir in inhibition of viral replication. Together, these results demonstrate an unexpected promiscuity of the viral RNA polymerase and provide a basis for repurpose and design of nucleotide analog drugs for COVID-19.

The effect of heat on SARS-CoV-2 viability and RNA integrity as determined by plaque assay, virus culture and real-time RT-PCR (preprint)

The effect of heat on SARS-CoV-2/England/2/2020 viability was assessed by plaque assay and virus culture. Heating to 56{degrees}C and 60{degrees}C for 15, 30 and 60 minutes led to a reduction in titre of between 2.1 and 4.9 log 10 pfu/ml but complete inactivation was not observed. At 80{degrees}C plaques were observed after 15 and 30 minutes of heating, however after 60 minutes viable virus was only detected following virus culture. Heating to 80{degrees}C for 90 minutes and 95{degrees}C for 1 and 5 minutes resulted in no viable virus being detected. At 56{degrees}C and 60{degrees}C significant variability between replicates was observed and the titre often increased with heat-treatment time. Nucleic acids were extracted and tested by RT-PCR. Sensitivity of the RT-PCR was not compromised by heating to 56{degrees}C and 60{degrees}C. Heating to 80{degrees}C for 30 minutes or more and 95{degrees}C for 1 or 5 minutes however, resulted in an increase of at least three Ct values. This increase remained constant when different dilutions of virus underwent heat treatment. This indicates that high temperature heat inactivation of clinical samples prior to nucleic acid extraction could significantly affect the ability to detect virus in clinical samples from patients with lower viral loads by RT-PCR.

Photocatalyst under visible light irradiation inactivates SARS-CoV-2 on an abiotic surface (preprint)

There is a worldwide attempt to develop prevention strategies against SARS-CoV-2 transmission. Here we examined the effectiveness of visible light-responsive photocatalyst RENECAT on the inactivation of SARS-CoV-2 under different temperatures and exposure durations. The viral activation on the photocatalyst-coated glass slides decreased from 5.93{mp}0.38 logTCID50/ml to 3.05{mp} 0.25 logTCID50/ml after exposure to visible light irradiation for 6h at 20 degree C. On the other hand, lighting without the photocatalyst, or the photocatalyst-coat without lighting retained viral stability. Immunoblotting and electron microscopic analyses showed the reduced amounts of spike protein on the viral surface after the photocatalyst treatment. Our data suggest a possible implication of the photocatalyst on the decontamination of the SARS-CoV-2 in indoor environments, thereby preventing indirect viral spread.

Genetic diversity analysis of the D614G mutation in SARS-CoV-2 (preprint)

In this work, we evaluated the levels of genetic diversity in 18 genomes of SARS-CoV-2 carrying the D614G mutation, coming from Malaysia and Venezuela and publicly available at the National Center of Biotechnology and Information (NCBI). These haplotypes were previously used for phylogenetic analysis, following the LaBECom protocols. All gaps and unconserved sites were extracted for the construction of a phylogenetic tree. As specific methodologies for paired FST estimators, Molecular Variance (AMOVA), Genetic Distance, mismatch, demographic and spatial expansion analyses, molecular diversity and evolutionary divergence time analyses, 20,000 random permutations were always used. The results revealed the presence of only 57 sites of polymorphic and parsimonium-informative among the 29,827bp analyzed and the analyses based on FST values confirmed the presence of two distinct genetic entities with fixation index of 22% and with a higher component of population variation (78.14%). Tau variations revealed a significant time of divergence, supported by mismatch analysis of the observed distribution ({tau} = 42%). It is safe to say that the small number of existing polymorphisms should not reflect major changes in the protein products of viral populations in both countries and this consideration provides the safety that, although there are differences in the haplotypes studied, these differences are minimal for both regions analyzed geographically and, therefore, it seems safe to extrapolate the levels of polymorphism and molecular diversity found in the samples for other mutant genomes of SARS-CoV-2 in other countries. This reduces speculation about the possibility of large differences between mutant strains of SARS-CoV-2 (D614G) and wild strains, at least at the level of their protein products, although the mutant form has higher transmission speed and infection. The analyses suggest that possible variations in protein products, of the wild virus in relation to its mutant form, should be minimal, bringing peace of mind as to the increased risk of death from the new form of the virus, as well as possible problems of gradual adjustments in some molecular targets for vaccines.

Temporal patterns in the evolutionary genetic distance of SARS-CoV-2 during the COVID-19 pandemic (preprint)

Background: During the pandemic of coronavirus disease 2019 (COVID-19), the genetic mutations occurred in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cumulatively or sporadically. In this study, we employed a computational approach to identify and trace the emerging patterns of the SARS-CoV-2 mutations, and quantify accumulative genetic distance across different periods and proteins. Methods: Full-length human SARS-CoV-2 strains in United Kingdom were collected. We investigated the temporal variation in the evolutionary genetic distance defined by the Hamming distance since the start of COVID-19 pandemic. Findings: Our results showed that the SARS-CoV-2 was in the process of continuous evolution, mainly involved in spike protein (S protein), the RNA-dependent RNA polymerase (RdRp) region of open reading frame 1 (ORF1) and nucleocapsid protein (N protein). By contrast, mutations in other proteins were sporadic and genetic distance to the initial sequenced strain did not show an increasing trend.

D614G substitution enhances the stability of trimeric SARS-CoV-2 spike protein (preprint)

SARS-CoV-2 spike protein with D614G substitution has become the dominant variant in the ongoing COVID-19 pandemic. Several studies to characterize the new virus expressing G614 variant show that it exhibits increased infectivity compared to the ancestral virus having D614 spike protein. Here, using in-silico mutagenesis and energy calculations, we analyzed inter-residue interaction energies and thermodynamic stability of the dominant (G614) and the ancestral (D614) variants of spike protein trimer in closed and partially open conformations. We find that the local interactions mediated by aspartate at the 614th position are energetically frustrated and create unfavourable environment. Whereas, glycine at the same position confers energetically favourable environment and strengthens intra- as well as inter-protomer association. Such changes in the local interaction energies enhance the thermodynamic stability of the spike protein trimer as free energy difference ({Delta}{Delta}G) upon glycine substitution is -2.6 kcal/mol for closed conformation and -2.0 kcal/mol for open conformation. Our results on the structural and energetic basis of enhanced stability hint that G614 may confer increased availability of functional form of spike protein trimer and consequent in higher infectivity than the D614 variant.

Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19 (preprint)

The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 Å cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19.

Structure Basis for Inhibition of SARS-CoV-2 by the Feline Drug GC376 (preprint)

The pandemic of SARS-CoV-2 coronavirus disease-2019 (COVID-19) caused by SARS-COV-2 continues to ravage many countries in the world. Mpro is an indispensable protein for viral translation in SARS-CoV-2 and a potential target in high-specificity anti-SARS-CoV-2 drug screening. In this study, to explore potential drugs for treating COVID-19, we elucidated the structure of SARS-CoV-2 Mpro and explored the interaction between Mpro and GC376, an antiviral drug used to treat a range of coronaviruses in Feline via inhibiting Mpro. The availability and safety of GC376 were proved by biochemical and cell experiments in vitro. We determined the structure of an important protein, Mpro, in SARS-CoV-2, and revealed the interaction of GC376 with the viral substrate and inhibition of the catalytic site of SARS-CoV-2 Mpro.

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir (preprint)

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 [A]. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.