Structure and proposed DNA delivery mechanism of a marine roseophage.

Tailed bacteriophages (order, Caudovirales) account for the majority of all phages. However, the long flexible tail of siphophages hinders comprehensive investigation of the mechanism of viral gene delivery. Here, we report the atomic capsid and in-situ structures of the tail machine of the marine siphophage, vB_DshS-R4C (R4C), which infects Roseobacter. The R4C virion, comprising 12 distinct structural protein components, has a unique five-fold vertex of the icosahedral capsid that allows genome delivery. The specific position and interaction pattern of the tail tube proteins determine the atypical long rigid tail of R4C, and further provide negative charge distribution within the tail tube. A ratchet mechanism assists in DNA transmission, which is initiated by an absorption device that structurally resembles the phage-like particle, RcGTA. Overall, these results provide in-depth knowledge into the intact structure and underlining DNA delivery mechanism for the ecologically important siphophages.

A Hemagglutinin Stem Vaccine Designed Rationally by AlphaFold2 Confers Broad Protection against Influenza B Infection.

Two lineages of influenza B viruses (IBV) co-circulating in human beings have been posing a significant public health burden worldwide. A substantial number of broadly neutralizing antibodies (bnAbs) have been identified targeting conserved epitopes on hemagglutinin (HA) stem domain, posing great interest for universal influenza vaccine development. Various strategies to design immunogens that selectively present these conserved epitopes are being explored. However, it has been a challenge to retain native conformation of the HA stem region, especially for soluble expression in prokaryotic systems. Here, using a structure prediction tool AlphaFold2, we rationally designed a stable stem antigen "B60-Stem-8071", an HA stem vaccine derived from B/Brisbane/60/2006 grafted with a CR8071 epitope as a linker. The B60-Stem-8071 exhibited better solubility and more stable expression in the E. coli system compared to the naïve HA stem antigen. Immunization with B60-Stem-8071 in mice generated cross-reactive antibodies and protected mice broadly against lethal challenge with Yamagata and Victoria lineages of influenza B virus. Notably, soluble expression of B60-stem-8071 in the E. coli system showed the potential to produce the influenza B vaccine in a low-cost way. This study represents a proof of concept for the rational design of HA stem antigen based on structure prediction and analysis.

A stepwise docking molecular dynamics approach for simulating antibody recognition with substantial conformational changes.

Conformational changes or rearrangements are common events during inter-biomolecular recognition. Tracking these changes are essential for exploring the allosteric mechanism and it is usually achieved by molecular dynamics simulation in silico. We previously identified a broad-neutralizing antibody against H5 influenza virus, 13D4, and solved the crystal structures of the free 13D4 Fab and its complex with hemagglutinin (HA). Structural comparison of the unbound and bound 13D4 Fabs showed that the heavy chain complementarity-determining region 3 (HCDR3) undergoes a substantial conformational rearrangement when it recognizes the receptor-binding site (RBS). Here, we used molecular dynamics (MD) to simulate the conformational changes that occur during antibody recognition. We showed that neither conventional MD nor steered MD could recapitulate the loop fitting of the RBS structure contour. Consequently, to simulate these challenging conformational changes, we engaged a stepwise docking MD method that allowed for the gradual docking of the ligand to receptor. This new method recapitulates the bound shape of the HCDR3 and provides the best approximation of the shape rendered by the co-crystal structure, with an RMSD of 0.926 Å. This strategy affords a flexible MD approach for simulating complicated conformational changes that occur during molecular recognition, and helps to provide an understanding of the involved allosteric mechanism.