Leveraging vaccination-induced protective antibodies to define conserved epitopes on influenza N2 neuraminidase.

There is growing appreciation for neuraminidase (NA) as an influenza vaccine target; however, its antigenicity remains poorly characterized. In this study, we isolated three broadly reactive N2 antibodies from the plasmablasts of a single vaccinee, including one that cross-reacts with NAs from seasonal H3N2 strains spanning five decades. Although these three antibodies have diverse germline usages, they recognize similar epitopes that are distant from the NA active site and instead involve the highly conserved underside of NA head domain. We also showed that all three antibodies confer prophylactic and therapeutic protection in vivo, due to both Fc effector functions and NA inhibition through steric hindrance. Additionally, the contribution of Fc effector functions to protection in vivo inversely correlates with viral growth inhibition activity in vitro. Overall, our findings advance the understanding of NA antibody response and provide important insights into the development of a broadly protective influenza vaccine.

Atomic model of a cypovirus built from cryo-EM structure provides insight into the mechanism of mRNA capping.

The cytoplasmic polyhedrosis virus (CPV) from the family Reoviridae belongs to a subgroup of "turreted" reoviruses, in which the mRNA capping activity occurs in a pentameric turret. We report a full atomic model of CPV built from a 3D density map obtained using cryoelectron microscopy. The image data for the 3D reconstruction were acquired exclusively from a CCD camera. Our structure shows that the enzymatic domains of the pentameric turret of CPV are topologically conserved and that there are five unique channels connecting the guanylyltransferase and methyltransferase regions. This structural organization reveals how the channels guide nascent mRNA sequentially to guanylyltransferase, 7-N-methyltransferase, and 2'-O-methyltransferase in the turret, undergoing the highly coordinated mRNA capping activity. Furthermore, by fitting the deduced amino acid sequence of the protein VP5 to 120 large protrusion proteins on the CPV capsid shell, we confirmed that this protrusion protein is encoded by CPV RNA segment 7.

Genome organization in double-stranded DNA viruses observed by cryoET.

Double-stranded DNA (dsDNA) viruses package their genetic material into protein cages with diameters usually a few hundred times smaller than the length of their genome. Compressing the relatively stiff and highly negatively charged dsDNA into a small volume is energetically costly and mechanistically enigmatic. Multiple models of dsDNA packaging have been proposed based on various experimental evidence and simulation methods, but direct observation of any viral genome organization is lacking. Here, using cryoET and an improved data processing scheme that utilizes information from the encaging protein shell, we present 3D views of dsDNA genome inside individual viral particles at resolution that densities of neighboring DNA duplexes are readily separable. These cryoET observations reveal a "rod-and-coil" fold of the dsDNA that is conserved among herpes simplex virus type 1 (HSV-1) with a spherical capsid, bacteriophage T4 with a prolate capsid, and bacteriophage T7 with a proteinaceous core inside the capsid. Finally, inspired by the genome arrangement in partially packaged T4 particles, we propose a mechanism for the genome packaging process in dsDNA viruses.

High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike.

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we establish a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrates a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, we apply this method to a region in the S2 domain that includes the first heptad repeat and central helix. Our results reveal that besides K986P and V987P, several mutations simultaneously improve expression and significantly lower the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.

High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike.

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we established a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrated a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, this method was applied to a region in the S2 domain that includes the first heptad repeat and central helix. Our results revealed that besides K986P and V987P, several mutations simultaneously improved expression and significantly lowered the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.