Biology subjects

Miller, E. H.

Publications and source records attributed to Miller, E. H..

5 recordsLinked to original sources

bioRxiv2025

Structural Dynamics and Allosteric Communication of a SARS-Like Bat Coronavirus Spike Glycoprotein

SARS-like bat coronaviruses (CoVs) pose ongoing public health risks due to their zoonotic potential, making it important to understand the molecular pathways driving their evolution. We recently showed that SHC014-CoV can infect human cell lines in an ACE2-dependent manner after acquiring two spike ectodomain mutations (F294L and A835D). However, how the wild-type (WT) SHC014 spike differs dynamically from these mutants remains unclear. Here, we built fully glycosylated ectodomain models of WT and three mutants (F294L, A835D, and the double mutant, DM) and performed triplicate 1-s all-atom molecular dynamics (MD) simulations for each variant. The two mutations exhibit epistasis, altering structural rearrangements relative to WT. Notably, the DM receptor binding domain (RBD) begins sampling the open conformation in our conventional MD. At the atomic level, the DM spike mitigates the dense negative packing introduced by A835D through a salt-bridge network, while F294L disrupts {pi}-mediated interactions, together enhancing RBD opening propensity--critical for viral entry. Increased flexibility of the subdomain-2 "620-loop" further modulates DM RBD openness. Dynamical network analysis identified three allosteric communication pathways. In WT and F294L, "Pathway 1" forms the baseline route linking the 620-loop to the RBD, whereas in A835D and DM it extends to the FPPR, reshaping long-range communication. "Pathway 2" is conserved across variants but is most prominent in WT and F294L. "Pathway 3" appears only in A835D and DM, compensating for reduced communication along Pathway 2. Overall, this work provides an atomistic perspective on SHC014 molecular adaptation during host-to-host transmission and highlights mechanistic features that may inform future therapeutic and pandemic-preparedness efforts. Statement of SignificanceBat coronaviruses are an important source of future pandemic threats, but we still know little about how small genetic changes help them infect humans. In this study, we used detailed computer simulations to watch how tiny mutations in a bat coronavirus spike protein change its motion and shape. We found that two specific mutations work together to make the spike more likely to open--a step required for the virus to enter human cells. By revealing how these molecular changes increase infection potential, our work helps improve understanding of coronavirus evolution and may guide strategies to prepare for future outbreaks.

biophysics↗

bioRxiv2025

Bat sarbecovirus WIV1-CoV bears an adaptive mutation that alters spike dynamics and enhances ACE2 binding

SARS-like betacoronaviruses (sarbecoviruses) endemic in bats pose a significant zoonotic threat to humans. Genetic pathways associated with spillover of bat sarbecoviruses into humans are incompletely understood. We previously showed that the WT spike of the rhinolophid bat coronavirus SHC014-CoV has poor entry activity and uncovered two distinct genetic pathways outside the receptor-binding domain (RBD) that increased spike opening, ACE2 binding, and cell entry. Herein, we show that the widely studied bat sarbecovirus WIV1-CoV is likely a cell culture-adapted variant of Rs3367-CoV, which was sequenced from the same population of rhinolophid bats as SHC014-CoV. We demonstrate that the acquisition of a single amino-acid substitution in the 630 loop of the S1 subunit was the key spike adaptation event during the successful isolation of WIV1-CoV, and that it enhances spike opening, virus-receptor recognition, and cell entry in much the same manner as the substitutions we previously identified in SHC014-CoV using a pseudotype system. The conformational constraints on both the SHC014-CoV and Rs3367-CoV spikes could be alleviated by pre-cleaving them with trypsin, suggesting that the spike-opening substitutions arose to circumvent the lack of S1-S2 cleavage. We propose that the locked-down nature of these spikes and their requirement for S1-S2 cleavage to engage ACE2 represent viral optimizations for a fecal-oral lifestyle and immune evasion in their natural hosts. These adaptations may be a broader property of bat sarbecoviruses than currently recognized. The acquisition of a polybasic furin cleavage site at the S1-S2 boundary is accepted as a key viral adaptation for SARS-CoV-2 emergence that overcame a host protease barrier to viral entry in the mammalian respiratory tract. Our results suggest alternative spillover scenarios in which spike-opening substitutions that promote virus-receptor binding and entry could precede, or even initially replace, substitutions that enhance spike cleavage in the zoonotic host. Author SummaryRecent epidemic-causing coronaviruses, including SARS-CoV-2, originated in bats. Large numbers of such viruses circulate in bats and pose clear and present risks to humans. However, our incomplete understanding of the variables that influence viral spillover into new hosts challenges attempts to stratify viruses by threat level. We showed previously that the entry spike of the bat coronavirus SHC014-CoV, closely related to SARS-CoV-1 and SARS-CoV-2, exists largely in a closed conformation that is incompatible with its binding to the viral receptor, ACE2, and that genetic changes in key control sequences open the spike and unlock its entry activity. Here, we extend these findings to a second bat coronavirus. We demonstrate that WIV1-CoV, a highly studied virus that was previously isolated from the same population of Chinese horseshoe bats, is in fact a variant of the bat coronavirus Rs3367-CoV that acquired a genetic change in the same hotspot region of the spike during its propagation in cell culture. Our findings support the idea that the closed spikes of at least some (and likely, many) bat coronaviruses, while exquisitely adapted to their natural milieu in the bat digestive system, suffer poor functional activity outside this milieu, imposing a barrier to viral spillover that must be overcome through viral adaptation. Further, we identify spike genetic changes that overcome this deficit and may have value as prognostic markers of zoonotic risk.

microbiology↗

bioRxiv2024

Epistasis between N-terminal and receptor-binding domains drives cell entry in a bat coronavirus spike

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles heels that could be targeted with countermeasures.

microbiology↗

bioRxiv2023

Simulation-Driven Design of Stabilized SARS-CoV-2 Spike S2 Immunogens

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimers opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

biophysics↗

bioRxiv2022

Vocal traits of shorebird chicks are related to body mass and sex

Acoustic communication is critical during early life phases in precocial birds. For example, adult alarm calls can elicit antipredator behaviour in young, and chick vocalisations can communicate information to parents about chick identity, condition, location, sex, or age. We investigated whether chick calls of two species of Australian Charadriidae vary with sex or body mass. We handled Red-capped Plover Charadrius ruficapillus and Southern Masked Lapwing Vanellus miles novaehollandiae chicks for purposes of measurement, blood sampling, and banding. We opportunistically recorded their distress calls while in the hand, and analysed the calls to determine whether call structure is related to sex or body mass (a proxy for age). We measured five traits per call, plus time intervals between successive calls, for 26 plover chicks (2600 calls) and 95 lapwing chicks (6835 calls). In plovers, inter-call intervals were shorter in males and both inter-call interval and the dominant frequency range of calls decreased with increasing body mass. In lapwings, frequency modulation (computed as the range in the rate of change of the dominant frequency) was lower in male calls. The dominant frequency range of lapwing calls decreased with mass in both sexes, but the decline was greater in males, resulting in a lower dominant frequency range in males. Frequency modulation and entropy of lapwing calls also decreased with increasing body mass. Minimum dominant frequency did not change with body mass or sex in either species. Our study provides the first evidence for charadriid chicks of (a) a sexual difference in call structure and rate and (b) gradual growth-related changes in call structure and rate, across chicks. Studies on calls from a greater range of chick ages and from more species within this large and diverse family would be valuable. We provide a foundation for further studies of shorebird vocalisations during growth, which may elucidate the development and functional significance of such vocalisations.

animal behavior and cognition↗