25 Recently a novel coronavirus (2019-nCoV) has emerged from Wuhan, China, 26 causing symptoms in humans similar to those caused by SARS coronavirus (SARS-27 CoV). Since SARS-CoV outbreak in 2002, extensive structural analyses have revealed 28 key atomic-level interactions between SARS-CoV spike protein receptor-binding domain 29 (RBD) and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate 30 both the cross-species and human-to-human transmissions of SARS-CoV. Here we 31 analyzed the potential receptor usage by 2019-nCoV, based on the rich knowledge about 32 SARS-CoV and the newly released sequence of 2019-nCoV. First, the sequence of 2019-33 nCoV RBD, including its receptor-binding motif (RBM) that directly contacts ACE2, is 34 similar to that of SARS-CoV, strongly suggesting that 2019-nCoV uses ACE2 as its 35 receptor. Second, several critical residues in 2019-nCoV RBM (particularly Gln493) 36 provide favorable interactions with human ACE2, consistent with 2019-nCoV's capacity 37 for human cell infection. Third, several other critical residues in 2019-nCoV RBM 38 (particularly Asn501) are compatible with, but not ideal for, binding human ACE2, 39 suggesting that 2019-nCoV has acquired some capacity for human-to-human 40 transmission. Last, while phylogenetic analysis indicates a bat origin of 2019-nCoV, 41 2019-nCoV also potentially recognizes ACE2 from a diversity of animal species (except 42 mice and rats), implicating these animal species as possible intermediate hosts or animal 43 models for 2019-nCoV infections. These analyses provide insights into the receptor 44 usage, cell entry, host cell infectivity and animal origin of 2019-nCoV, and may help 45 epidemic surveillance and preventive measures against 2019-nCoV. 46 47 on March 16, 2020 by guest http://jvi.asm.org/ Downloaded from Significance 48 The recent emergence of Wuhan coronavirus (2019-nCoV) puts the world on 49 alert. 2019-nCoV is reminiscent of the SARS-CoV outbreak in 2002-2003. Our decade-50 long structural studies on the receptor recognition by SARS-CoV have identified key 51 interactions between SARS-CoV spike protein and its host receptor angiotensin-52 converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-53 human transmissions of SARS-CoV. One of the goals of SARS-CoV research was to 54 build an atomic-level iterative framework of virus-receptor interactions to facilitate 55 epidemic surveillance, predict species-specific receptor usage, and identify potential 56 animal hosts and animal models of viruses. Based on the sequence of 2019-nCoV spike 57 protein, we apply this predictive framework to provide novel insights into the receptor 58 usage and likely host range of 2019-nCoV. This study provides a robust test of this 59 reiterative framework, providing the basic, translational and public health research 60 communities with predictive insights that may help study and battle this novel 2019-61 nCoV. 62 63 on March 16, 2020 by guest

Emerging viral infections are difficult to control as heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. SARS-CoV and MERS-CoV successively emerged causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. Here we show that a nucleotide prodrug GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat-CoVs, prepandemic bat-CoVs and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory functions. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and possibly most importantly, emerging CoV of the future.

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.

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-N4-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-N4-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.

The spike D614G substitution is prevalent in global SARS-CoV-2 strains, but its effects on viral pathogenesis and transmissibility remain unclear. We engineered a SARS-CoV-2 variant containing this substitution. The variant exhibits more efficient infection, replication, and competitive fitness in primary human airway epithelial cells, but maintains similar morphology and in vitro neutralization properties, compared with the ancestral wild-type virus. Infection of human angiotensin-converting enzyme 2 (ACE2) transgenic mice and Syrian hamsters with both viruses resulted in similar viral titers in respiratory tissues and pulmonary disease. However, the D614G variant transmits significantly faster and displayed increased competitive fitness than the wild-type virus in hamsters. These data show that the D614G substitution enhances SARS-CoV-2 infectivity, competitive fitness, and transmission in primary human cells and animal models.