Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the ...mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Matrix metalloproteinases (MMPs) are extracellular proteases highly expressed at wound sites. However, the precise function of MMPs during reepithelialization in vivo has been elusive in mammalian ...models because of the high level of redundancy among the 24 mammalian MMPs. For this reason we used Drosophila melanogaster, whose genome encodes only two MMPs-one secreted type (Mmp1) and one membrane-anchored type (Mmp2)-to study the function and regulation of the secreted class of MMPs in vivo. In the absence of redundancy, we found that the Drosophila secreted MMP, Mmp1, is required in the epidermis to facilitate reepithelialization by remodeling the basement membrane, promoting cell elongation and actin cytoskeletal reorganization, and activating extracellular signal-regulated kinase signaling. In addition, we report that the jun N-terminal kinase (JNK) pathway upregulates Mmp1 expression after wounding, but that Mmp1 is expressed independent of the JNK pathway in unwounded epidermis. When the JNK pathway is ectopically activated to overexpress Mmp1, the rate of healing is accelerated in an Mmp1-dependent manner. A primary function of Mmp1, under the control of the JNK pathway, is to promote basement membrane repair, which in turn may permit cell migration and the restoration of a continuous tissue.
Thirty-four adults received two 100-μg injections of Moderna’s mRNA SARS-CoV-2 vaccine, and serum anti–spike protein and neutralizing antibody titers were measured at day 119 — 90 days after the ...second injection. By three different assays, binding and neutralizing antibody titers declined slightly but remained elevated and higher than titers in convalescent plasma.
Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show ...that the ribonucleoside analog β-d-N
-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N
-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development ...of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity
. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant
SARS-CoV-2 as well as CD8
T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for ...broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 μM). Weaker activity is observed in Vero E6 cells (EC50 = 1.65 μM) because of their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase of SARS-CoV-2. In mice infected with the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with vehicle-treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.
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•Remdesivir binding of active site of polymerase is conserved across all human CoVs•Remdesivir inhibits SARS-CoV-2 in primary and continuous human lung cell cultures•Remdesivir potency depends on cell-type-specific metabolism to its active form•Therapeutic remdesivir reduces viral loads and improves outcomes in mice
SARS-CoV-2 causes severe lung disease (COVID-19) in humans. Pruijssers et al. demonstrate that the antiviral drug remdesivir potently inhibits SARS-CoV-2 in human lung cell cultures. Therapeutic treatment of infected mice with remdesivir reduces viral loads and improves clinical outcomes, further supporting use of remdesivir for the treatment of COVID-19.
The nucleoside analog remdesivir (RDV) is a Food and Drug Administration-approved antiviral for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Thus, it is ...critical to understand factors that promote or prevent RDV resistance. We passaged SARS-CoV-2 in the presence of increasing concentrations of GS-441524, the parent nucleoside of RDV. After 13 passages, we isolated three viral lineages with phenotypic resistance as defined by increases in half-maximal effective concentration from 2.7- to 10.4-fold. Sequence analysis identified nonsynonymous mutations in nonstructural protein 12 RNA-dependent RNA polymerase (
-RdRp): V166A, N198S, S759A, V792I, and C799F/R. Two lineages encoded the S759A substitution at the RdRp Ser
-Asp-Asp active motif. In one lineage, the V792I substitution emerged first and then combined with S759A. Introduction of S759A and V792I substitutions at homologous
positions in murine hepatitis virus demonstrated transferability across betacoronaviruses; introduction of these substitutions resulted in up to 38-fold RDV resistance and a replication defect. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a roughly 10-fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, whereas
-V792I diminished the uridine triphosphate concentration needed to overcome template-dependent inhibition associated with RDV. The in vitro-selected substitutions identified in this study were rare or not detected in the greater than 6 million publicly available
-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in clinical settings.
It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread ...propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.
Synopsis
The DnaJC5 protein, cysteine string protein‐α (CSPα), stimulates the release of neurodegenerative proteins through a non‐canonical exocytosis pathway.
Increased expression of DnaJC5 promotes extracellular release of the microtubule‐associated protein tau, α‐synuclein, and TAR‐DNA binding protein.
Release of these proteins is dependent upon both the SNARE, SNAP‐23, and the constitutively expressed Hsp70 variant, Hsc70.
This function of DnaJC5 and the Hsc70 complex is unique as other DnaJs and Hsp70 variants do not promote this mechanism.
This finding provides a mechanism for how aggregate‐prone proteins can exit the cell and possibly seed propagation.
DnaJC5 is a co‐chaperone for Hsc70 and SNARE‐dependent neuronal release of disease proteins such as tau, TDP‐43, or α‐synuclein.
Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, ...we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies.
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•Structure-guided design generates MERS-CoV spike stabilized stem (SS) antigens•Cross-reactive CoV-spike-stem-specific monoclonal antibodies•Structures of the Fab22-spike complexes reveal a conserved, protective epitope•Passive transfer of IgG22 protects mice against MERS-CoV and SARS-CoV-2 challenge
Hsieh et al. generate MERS-CoV spike stabilized stem (SS) antigens using a structure-guided approach. Mice immunized with MERS SS are protected against MERS-CoV challenge and used for isolation of cross-reactive monoclonal antibodies, including IgG22, which protects mice against MERS-CoV and SARS-CoV-2 challenges. Structures of Fab22-spike complexes reveal a conserved epitope.
Tylosis esophageal cancer (TOC) is an autosomal-dominant syndrome characterized by palmoplantar keratoderma, oral precursor lesions, and a high lifetime risk of esophageal cancer. We have previously ...localized the TOC locus to a small genomic interval within chromosomal region 17q25. Using a targeted capture array and next-generation sequencing, we have now identified missense mutations (c.557T>C p.Ile186Thr and c.566C>T p.Pro189Leu in RHBDF2, which encodes the inactive rhomboid protease RHBDF2 (also known as iRhom2), as the underlying cause of TOC. We show that the distribution of RHBDF2 in tylotic skin is altered in comparison with that in normal skin, and immortalized tylotic keratinocytes have decreased levels of total epidermal growth factor receptor (EGFR) and display an increased proliferative and migratory potential relative to normal cells, even when normal cells are stimulated with exogenous epidermal growth factor. It would thus appear that EGFR signaling is dysregulated in tylotic cells. Furthermore, we also show an altered localization of RHBDF2 in both tylotic and sporadic squamous esophageal tumors. The elucidation of a role of RHBDF2 in growth-factor signaling in esophageal cancer will help to determine whether targeting this pathway in chemotherapy for this and other squamous cell carcinomas will be effective.
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