Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined highresolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptoraccessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.coronavirus | neutralizing antibody | cryo-EM | X-ray crystallography | peplomer

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3=-5= exoribonuclease (ExoN). ␤-D-N 4 -Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC 50 ] ϭ 0.17 M) and Middle East respiratory syndrome CoV (MERS-CoV) (EC 50 ϭ 0.56 M) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virusmutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication. IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broadspectrum ribonucleoside analogue, ␤-D-N 4 -hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only lowlevel resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections. FIG 2 NHC inhibits MHV and MERS-CoV with minimal cytotoxicity. (A and B) Changes in MHV (A) and MERS-CoV (B) titers relative to vehicle control after treatment with increasing concentrations of NHC. The data represent the results of 6 independent experiments, each with 3 replicates. The error bars represent standard errors of the mean (SEM). (C) Changes in ti...

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic hereditary disorders in humans characterized by fluid-filled cysts, primarily in the kidneys. Cux1, a cell cycle regulatory gene highly expressed during kidney development, is elevated in the cyst-lining cells of Pkd1 mutant mice, and in human ADPKD cells. However, forced expression of Cux1 is insufficient to induce cystic disease in transgenic mice or to induce rapid cyst formation after cilia disruption in the kidneys of adult mice. Here we report a double mutant mouse model that has a conditional deletion of the Pkd1 gene in the renal collecting ducts together with a targeted mutation in the Cux1 gene (Pkd1;Cux1). While kidneys isolated from newborn Pkd1 mice exhibit cortical and medullary cysts, kidneys isolated from newborn Pkd1;Cux1 mice did not show any cysts. Because Cux1 are perinatal lethal, we evaluated Pkd1 mice that were heterozygote for the Cux1 mutation. Similar to the newborn Pkd1;Cux1 mice, newborn Pkd1;Cux1 mice did not show any cysts. Comparison of Pkd1 and Pkd1;Cux1 mice at later stages of development showed a reduction in the severity of PKD in the Pkd1;Cux1 mice. Moreover, we observed an increase in expression of the cyclin kinase inhibitor p27, a target of Cux1 repression, in the rescued collecting ducts. Taken together, our results suggest that Cux1 expression in PKD is not directly involved in cystogenesis but promotes cell proliferation required for expansion of existing cysts, primarily by repression of p27.

Alveolar soft-part sarcoma is a rare tumour. Patients commonly present with distant metastases both at the time of diagnosis and late in the course of disease. We present a case of pancreatic metastasis, occurring more than six years after diagnosis. Treatment consisted in subtotal pancreatoduodenectomy with pylorus resection. Both specific patterns of relapse and treatment opportunities of this uncommon feature are discussed.