This Version (2) corrects analysis that was based on the codon encoding Spike position 943; the apparent mutation at 943 was the result of a sequence error. The main conclusions of the paper regarding the mutation in Spike at 614 and recombination still hold. The key difference in version 2 is that we have removed the original figure 6, which was based on the 943 sequencing artifact, and instead moved a figure illustrating recombination that was independent of position 943 from the supplement into the main text. BK SummaryWe have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified thirteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; it began spreading in Europe in early February, and when introduced to new regions it rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.

COVID-19 has become a global pandemic caused by the novel coronavirus SARS-CoV-2. Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis, discovering new drugs, and developing a vaccine. We show evidence of strong purifying selection around the receptor binding motif (RBM) in the spike and other genes among bat, pangolin, and human coronaviruses, suggesting similar evolutionary constraints in different host species. We also demonstrate that SARS-CoV-2’s entire RBM was introduced through recombination with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2’s ability to infect humans. Similar purifying selection in different host species, together with frequent recombination among coronaviruses, suggests a common evolutionary mechanism that could lead to new emerging human coronaviruses.

Heterodyned femtosecond infrared two-pulse and three-pulse photon echoes of the dipeptide acetylproline-NH 2 in D 2 O and CDCl 3 have been measured and the results have been compared with force field calculations of the peptide structures. The heterodyned two-dimensional infrared (2D IR) spectra obtained from the measurements exhibit diagonal peaks and cross-peaks that are determined by the structures and vibrational dynamics of the acetylproline-NH 2 molecule. The two-pulse measurements are analogous to 2D COSY experiments in NMR spectroscopy. In CDCl 3 , the 2D IR spectra from the two-pulse experiments resolve two acetyl amide I bands and two amino amide I bands that are not resolved in the linear spectrum. Thus, acetylproline-NH 2 must have at least two structures in CDCl 3 . The angles between the amide I transition dipoles of the structures were determined to be <20°and 35°from polarized 2D IR measurements. A single structure is found in D 2 O with an angle of <20°. The infrared analogue to 2D NMR NOESY experiments has also been performed utilizing three-pulse photon echoes. In these three-pulse experiments, the cross-peak intensities and polarizations are found to change with the waiting time between the second and third pulses. By comparison to the changes in the diagonal peaks, these effects are attributed to population and coherence transfer processes. The vibrational dynamics and inhomogeneous distributions at the acetyl and amino ends of acetylproline-NH 2 are observed to be different. † Part of the special issue "Bruce Berne Festschrift".

New experimental techniques are capable of determining the relative population of conformations adopted by short alanine peptides in water. Most of the existing all-atom force fields used to model proteins fail to reproduce the relative population of the most relevant conformations of peptides. The calculated relative population of conformations varies significantly depending on the force field chosen, thus urging the need to check the validity and consistency of force fields over a range of peptide lengths. Here, we show how the applicability of a modified version of AMBER force field (A94/MOD) can extend from short to large peptides. It is also capable of reproducing the expected shift in conformational preference with increasing peptide length and temperature. Importantly, the consistency of the force field is judged by direct comparison to experiments rather than to the relative energies of conformations obtained from ab initio calculations. Importantly, this study illustrates that many aspects of protein force fields are already well refined and may only require minor refinements to accurately reproduce experimental observations over a range of systems.

SummaryEliciting HIV-1-specific broadly neutralizing antibodies (bNAbs) remains a challenge for vaccine development, and the potential of passively delivered bNAbs for prophylaxis and therapeutics is being explored. We used neutralization data from four large virus panels to comprehensively map viral signatures associated with bNAb sensitivity, including amino acids, hypervariable region characteristics, and clade effects across four different classes of bNAbs. The bNAb signatures defined for the variable loop 2 (V2) epitope region of HIV-1 Env were then employed to inform immunogen design in a proof-of-concept exploration of signature-based epitope targeted (SET) vaccines. V2 bNAb signature-guided mutations were introduced into Env 459C to create a trivalent vaccine, and immunization of guinea pigs with V2-SET vaccines resulted in increased breadth of NAb responses compared with Env 459C alone. These data demonstrate that bNAb signatures can be utilized to engineer HIV-1 Env vaccine immunogens capable of eliciting antibody responses with greater neutralization breadth.