The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. Like other coronaviruses, the SARS-CoV-2 genome encodes spike (S) glycoproteins, which protrude from the surface of mature virions. The S glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. Surface location of the S glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. In the light of its crucial roles in viral infection and adaptive immunity, the S protein is the focus of most vaccine strategies as well as therapeutic interventions. In this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the SARS-CoV-2 S glycoprotein, aiming to provide valuable insights into the design and development of the S protein-based vaccines as well as therapeutics.

/pdf/the-sars-cov-2-spike-glycoprotein-biosynthesis-structure-3ntwvvyxm8.pdf

The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens.

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

/pdf/the-sars-cov-2-nucleocapsid-phosphoprotein-forms-mutually-6lugvmsj21.pdf

The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.

/pdf/sars-cov-2-tropism-entry-replication-and-propagation-1az1zoes19.pdf

SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery and development.

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein's C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.

/pdf/the-sars-cov-2-nucleocapsid-phosphoprotein-forms-mutually-xlbooh7x7i.pdf

The SARS-CoV-2 Nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.

A novel coronavirus has been reported as the causative pathogen of the Coronavirus disease 2019 (COVID-19) outbreak in Wuhan city, China in December 2019. Due to the rapid spread of the virus worldwide, it has been announced as a pandemic by the World Health Organization (WHO). Hospitalized patients in Wuhan were associated with the Huanan seafood wholesale market where live animals, such as poultry, bats, snakes, frogs, rabbits, marmots, and hedgehogs are sold in that market which suggests a possible zoonotic infection. It was suggested that bat is the natural host of SARS-CoV-2, but the intermediate host is still unclear. It is essential to identify the potential intermediate host to interrupt the transmission chain of the virus. Pangolin is a highly suspected candidate as an intermediate host for SARS-CoV-2. Recently, SARS-CoV-2 infection has been reported in cats, dogs, tigers, and lions. More recently SARS-CoV-2 infection affected minks severely and zoonotic transfer with a variant SARS-CoV-2 strain evidenced in Denmark, Netherlands, USA, and Spain suggesting animal-to-human and animal-to-animal transmission within mink farms. Furthermore, experimental studies documented the susceptibility of different animal species to SARS-CoV-2, such as mice, golden hamsters, cats, ferrets, non-human primates, and treeshrews. It is also essential to know the possibility of infection for other animal species. This short review aims to provide an overview on the relation between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and animals.

/pdf/an-overview-of-sars-cov-2-and-animal-infection-1flz9auof2.pdf

An Overview of SARS-CoV-2 and Animal Infection

We demonstrated that SF2523, a dual small molecule inhibitor of PI3K-α/mTOR/BRD4 pathways, can inhibit the replication of SARS-CoV-2 and its emerging variants of concern (VOCs), including Delta and Omicron. Further, we also found that SF2523 acts synergistically with remdesivir (RDV) and MU-UNMC-2 (a small molecule entry inhibitor of SARS-CoV-2).1 The ongoing COVID-19 pandemic due to the emergence of a novel coronavirus SARS-CoV-2 remains a significant health concern globally. Several vaccine candidates and anti-virals received emergency use authorization. However, these vaccines/anti-virals safety, efficacy and durability remain unknown, especially for the individuals with comorbid conditions, and VOCs, such as Delta, Omicron, BA.2 and Deltacron, which have evolved mutations in the receptor-binding domain of SARS-CoV-2 spike protein may even evade antibodies induced by vaccines or natural infection.2 Similarly, recently FDA-approved Molnupiravir and PAXLOVID remain sensitive towards VOCs.3 The in-depth understanding of the molecular mechanism of the SARS-CoV-2 lifecycle revealed the interaction of several host factors with viral proteins essential for the reproduction of progeny viruses, such as bromodomain, containing extra-terminal domain proteins (BETs), and the mTOR pathway.4 Recent studies identified 67 potential interactions between host and viral proteins essential for the SARS-CoV-2 lifecycle, like BRD2/BRD4 with the E protein of SARS-CoV-2,4 and suggested that BRD2 inhibition downregulates ACE2 expression, blocks the entry of SARS-CoV-2 into host cells and controls hyperactive immune response in COVID-19 patients through downregulation Interferon stimulated genes (ISGs).5 Targeted therapies that exploit host–virus interaction are likely to be least impacted by the VOCs of SARS-CoV-2 and are expected to produce more robust, durable treatment options. Therefore, we tested the anti-viral potential of SF2523 against the wild-type SARS-CoV-2 and VOCs. In UNCN1T (a bronchial epithelial cell line), Vero STAT1 KO and Calu-3 cells, the CC50 value of SF2523 is above 100 μM (Figure 1A,B; Figure S2A). SF2523 showed potent anti-viral activity with an IC50 of 1.52 μM (RDV IC50 of 1.06 μM) in UNCN1T cells (Figure 1C) and 1.02 μM (RDV IC50 of 1.03 μM) in Vero STAT1 KO cells (Figure 1D) at 24 hpi, respectively. At 48 hpi in UNCN1T cells, SF2523 has an IC50 of 1.58 μM (RDV IC50 of 2.75 μM) and in Vero STAT1 KO cells has an IC50 of 3.22 μM (RDV IC50 of .76 μM), respectively (Figure S1A; 1B). Similarly, in Calu-3 cells, SF2523 has an anti-viral activity with an IC50 of 2.08, 4.03, 0.86 and 4.03 μM against the Delta variant (linage: B.1.617.2; Figure 2A), the Omicron variant (linage: B.1.1.529; Figure 2B), the South African variant (linage: B.1.351; Figure 2C) and the Scotland variant (linage: B.1.222; Figure 2D), respectively. Since BRD4 promotes lung tissue fibrosis through an increase in the expression of pro-fibrotic genes, and the majority of severely ill COVID-19 patients suffer from pulmonary fibrosis,6 the inclusion of BRD4 inhibitor SF2523 in COVID-19 therapy will benefit patients to recover from lung fibrosis. This is further supported by a recent study showing that BET protein inhibition blocks cardiac irregularities and SARS-CoV-2 infection7 and may be useful to controlling the long COVID-19 effects recently seen in many recovered patients. Combining multiple drugs with different modes of action has synergistic effects and has been used as anti-virals. Therefore, we evaluated a series of fixed-dose combinations of SF2523 and RDV or MU-UNMC-2.1 The combined doses of SF2523/RDV and SF2523/MU-UNMC-2 have CC50 values above 100 μM (Figure S2B and C). In UNCN1T cells at 24 hpi, when a fixed dose of 0.1 μM SF2523 is combined with different dosages of RDV, it has an IC50 value of 0.62 μM (Figure 3A) that is lower than individual IC50 of both the compounds. The dose–response percent inhibition matrix of single and combination treatment of SF2523/RDV and SF2523/MU-UNMC-2 is described in Figure 3B and E. The 3D interaction landscape of SF2523/RDV and SF2523/MU-UNMC-2 was computed based on Loewe additive model using SynergyFinder v.2 (Figure 3C and F). The synergistic/antagonistic effects of drug combinations are reconfirmed using Chou and Talalay combination index (CI) theorem8 [CI < 1: synergism; = 1: additive effect; > 1: antagonism]. Using CompuSyn, we computed CI values of SF2523/RDV as CI < 1 and CI of SF2523/MU-UNMC-2 as 0.129, confirming their synergistic effect. For SF2523/RDV, we obtained a dose reduction index (DRI) of 25.33 and 3.75 for SF2523 and RDV, and for SF2523/MU-UNMC-2, we obtained the DRI of 23.03 and 11.56 for SF2523 and MU-UNMC-2, respectively. This synergistic effect of SF2523 with RDV or MU-UNMC-2 is expected in a favourable shift in the plasma Cmax/EC90 ratio. Next, using NMR titration experiments by labelling 15N-labelled BRD4 domains, we showed that RDV is not able to bind to either BRD4-BD1, BRD4-BD2 or the ET domain of BRD4, as no significant chemical shift perturbations were observed in 1H,15N heteronuclear single quantum coherence spectra of these domains (Figure 4). This confirms that RDV does not alter the potency of SF2523 and does not interfere with the binding of SF2523 to BRD4. It is documented that SARS-CoV-2 infection in mammalian cells inhibits autophagy through various mechanisms, including the activation of autophagy inhibitory proteins (AKT1 and SKP2) and inhibition of proteins involved in autophagy initiation (AMPK, TSC2, Unc-51 Like Autophagy Activating Kinase 1 and Beclin 1), autophagosome formation (VPS34) and in membrane tethering and fusion of autophagosomes to endolysosomes (ATG14)9 and SARS-CoV-2, and its transmembrane protein ORF3a inhibits autophagy. Activating autophagy using small molecules that target PI3Kinase/Akt pathway or stabilize Beclin inhibits SARS-CoV-2 replication.9 Our studies suggest that SF2523 may be modulating the autophagy mechanisms (Figure S3) to limit SARS-CoV-2 infection similar to previously reported HIV-infected macrophages restrict infection through autophagy induction,10 although this warrants further in-depth investigation. In conclusion, we demonstrated that SF2523 could inhibit the replication of SARS-CoV-2 and its VOCs. Further, we found a synergistic effect of SF2523 with RDV or MU-UNMC-2 in a wide dose range. Therefore, we conclude that SF2523 (a PI3K-α/mTOR/BRD4 inhibitor) alone or combined with other anti-virials represents a future therapeutic approach to prevent the severe disease associated with SARS-CoV-2 and its VOCs. We acknowledge the UNMC BSL-3 core facility for allowing us to perform all in vitro experiments involving SARS-CoV-2. The University of Nebraska Medical Center BSL-3 Core Facility is administrated through the Office of the Vice-Chancellor for Research and supported by the Nebraska Research Initiative (NRI). The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: (a) SARS-Related Coronavirus 2; Isolate USA-WI1/2020 (NR-52384), (b) isolate hCoV-19/USA/MD-HP01542/2021 (NR-55282; B.1.351), (c) isolate hCoV-19/Scotland/CVR2224/2020 (NR-53945; B.1.222), (d) isolate hCoV-19/USA/MD-HP05647/202 (BEI; cat# NR-55672), (e) isolate hCoV-19/USA/HI-CDC-4359259-001/2021 (BEI; cat#NR-56475), (f) Quantitative PCR (qPCR) Control RNA from Heat-Inactivated SARS-Related Coronavirus 2, Isolate USA-WA1/2020, (NR 52347). This work is partially supported by the National Institute of Allergy and Infectious Diseases Grant R01 AI129745, 5P30 CA036727-33, Frances E. Lageschulte and Evelyn B. Weese New Frontiers in Medical Research Fund and independent research and development (IRAD) funding from the National Strategic Research Institute (NSRI) at the University of Nebraska to SNB and NIH grants HL151334 and CA252707 to T.G.K; and CA215651 to DLD. Fig S1. Wild type SARS-CoV-2 replication inhibition dose-response curves of SF2523 in UNCN1T and Vero STAT1 KO cells. (A, B). UNCN1T and Vero STAT1 knockout cells (20,000 cells/well) were seeded in 96-well plates 24 hours before infection. Different concentrations of SF2523 and remdesivir (100 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, 0.01 μM and 0.001 μM) were added to the cells 2 hours before infection. The cells were infected with 0.1 MOI of SARS-CoV-2 isolate USA-WI1/2020. Culture supernatant was collected at 48 hrs post-infection. The SARS-CoV-2 viral load was quantified in the culture supernatant using RT-QPCR with primer probes targeting E gene of SARS-CoV-2 using PrimeDirect Probe RT-qPCR Mix (TaKaRa Bio USA, Inc) and Applied Biosystems QuantStudio3 real-time PCR system (Applied Biosystems, Waltham, MA, USA) per the manufacturer's instructions. The SARS-CoV-2 genome equivalent copies were calculated using quantitative PCR (qPCR) control RNA from heat-inactivated SARS-CoV-2, isolate USA-WA1/2020 (BEI, Catalog# NR-52347). The percentage inhibition of SARS-CoV-2 replication in SF2523 and remdesivir treated wells was calculated with respect to virus concentration in positive control wells treated with DMSO (considered 0% inhibition) and negative control wells (uninfected cells). SF2523 (in blue) and remdesivir (in green) IC50 values were calculated using four-parameter variable slope sigmoidal dose-response models using Graph Pad Prism 8.0 software; [CC50: Drug concentration that required to reduces cell viability by 50%; IC50: Drug concentration that required to reduce the viral replication by 50%]. Fig S2. Cytotoxicity of SF2523 in Calu-3 cells (A) and combination of SF2523/RDV and SF2523/MU-UNMC-2 in UNCN1T cells (B and C) cells was measured by MTT assay. The cells were treated with increasing concentration of SF2523, SF2523/RDV and SF2523/MU-UNMC-2 (0.001 to 100 μM) and incubated at 37° C in a humidified 5% CO2 incubator. After 72-hour post-treatment, 20 μL of MTT substrate (5 mg/mL) was added to each well and incubated for 4 additional hours at 37° C in the dark. Then the culture media was carefully removed, and blue formazan crystals were dissolved in 200 °l of DMSO, and the purple color was read at 595 nm with a reference filter of 620 nm. In respective cells, SF2523, SF2523/RDV and SF2523/MU-UNMC-2 has a CC50 value above 100 °M. Fig S3. SF2523 may induces autophagy in SARS-CoV-2 infected Vero cells. The cells were treated with the indicated concentration of SF2523 and 5 mM 3-MA and infected with SARS-CoV-2. After 24h, cells were harvested, cell lysate prepared, total protein content quantified, and immunoblotting was performed. The upper and lower panels are the representative immunoblots of p62 and LC3B-I//II. Actin was used as a loading control. The upper and lower bar graphs show densitometric analysis of p62 and LC3B-II immunoblots, respectively normalized with actin. Data is expressed as mean ± SEM derived from two experiments. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ctm2.806

PI3K‐α/mTOR/BRD4 inhibitor alone or in combination with other anti‐virals blocks replication of SARS‐CoV‐2 and its variants of concern including Delta and Omicron

9-aminominocycline potentiates the efficacy of EIDD-1931 and PF-332 by targeting the papain like protease enzyme of SARS-CoV-2

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PI3K‐α/mTOR/BRD4 inhibitor alone or in combination with other anti‐virals blocks replication of SARS‐CoV‐2 and its variants of concern including Delta and Omicron

9-aminominocycline potentiates the efficacy of EIDD-1931 and PF-332 by targeting the papain like protease enzyme of SARS-CoV-2