Adaptation of SARS-CoV-2 to ACE2H353K mice reveals new spike residues that drive mouse infection.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.

A cell-free platform to measure coronavirus membrane fusion.

Here we present a protocol to measure coronavirus-mediated membrane fusion, an essential event in coronavirus cell entry. The approach uses nanoluciferase (Nluc) "HiBiT"-tagged corona virus-like particles (VLPs) and Nluc "LgBiT"-containing extracellular vesicles (EVs) as proxies for virus and cell, respectively. VLP-EV membrane fusion allows HiBiT and LgBiT to combine into measurable Nluc, which signifies virus fusion with target cell membranes. We highlight assay utility with methods to assess coronavirus-mediated fusion and its inhibition by antibodies and antiviral agents. For complete details on the use and execution of this protocol, please refer to Qing et al. (2021),1 Qing et al. (2022),2 and Marcink et al. (2022).3.

Dynamics of SARS-CoV-2 Spike Proteins in Cell Entry: Control Elements in the Amino-Terminal Domains.

Selective pressures drive adaptive changes in the coronavirus spike proteins directing virus-cell entry. These changes are concentrated in the amino-terminal domains (NTDs) and the receptor-binding domains (RBDs) of complex modular spike protein trimers. The impact of this hypervariability on virus entry is often unclear, particularly with respect to sarbecovirus NTD variations. Therefore, we constructed indels and substitutions within hypervariable NTD regions and used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-like particles and quantitative virus-cell entry assays to elucidate spike structures controlling this initial infection stage. We identified NTD variations that increased SARS-CoV-2 spike protein-mediated membrane fusion and cell entry. Increased cell entry correlated with greater presentation of RBDs to ACE2 receptors. This revealed a significant allosteric effect, in that changes within the NTDs can orient RBDs for effective virus-cell binding. Yet, those NTD changes elevating receptor binding and membrane fusion also reduced interdomain associations, leaving spikes on virus-like particles susceptible to irreversible inactivation. These findings parallel those obtained decades ago, in which comparisons of murine coronavirus spike protein variants established inverse relationships between membrane fusion potential and virus stability. Considerable hypervariability in the SARS-CoV-2 spike protein NTDs also appear to be driven by counterbalancing pressures for effective virus-cell entry and durable extracellular virus infectivity. These forces may selectively amplify SARS-CoV-2 variants of concern. IMPORTANCE Adaptive changes that increase SARS-CoV-2 transmissibility may expand and prolong the coronavirus disease 2019 (COVID-19) pandemic. Transmission requires metastable and dynamic spike proteins that bind viruses to cells and catalyze virus-cell membrane fusion. Using newly developed assays reflecting these two essential steps in virus-cell entry, we focused on adaptive changes in SARS-CoV-2 spike proteins and found that deletions in amino-terminal domains reset spike protein metastability, rendering viruses less stable yet more poised to respond to cellular factors that prompt entry and subsequent infection. The results identify adjustable control features that balance extracellular virus stability with facile virus dynamics during cell entry. These equilibrating elements warrant attention when monitoring the evolution of pandemic coronaviruses.

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles.

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.

Targeting Polyamines Inhibits Coronavirus Infection by Reducing Cellular Attachment and Entry.

Coronaviruses first garnered widespread attention in 2002 when the severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from bats in China and rapidly spread in human populations. Since then, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged and still actively infects humans. The recent SARS-CoV-2 outbreak and the resulting disease (coronavirus disease 2019, COVID19) have rapidly and catastrophically spread and highlighted significant limitations to our ability to control and treat infection. Thus, a basic understanding of entry and replication mechanisms of coronaviruses is necessary to rationally evaluate potential antivirals. Here, we show that polyamines, small metabolites synthesized in human cells, facilitate coronavirus replication and the depletion of polyamines with FDA-approved molecules significantly reduces coronavirus replication. We find that diverse coronaviruses, including endemic and epidemic coronaviruses, exhibit reduced attachment and entry into polyamine-depleted cells. We further demonstrate that several molecules targeting the polyamine biosynthetic pathway are antiviral in vitro. In sum, our data suggest that polyamines are critical to coronavirus replication and represent a highly promising drug target in the current and any future coronavirus outbreaks.

Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.