Changes in adenoviral chromatin organization precede early gene activation upon infection.

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

Multivalent display of engineered HIV-1 envelope trimers on silica nanoparticles for targeting and in vitro activation of germline VRC01 B cells.

Selective targeting of germline B cells with specifically designed germline-targeting HIV-1 envelope immunogens (GT-Env) is considered a feasible vaccination strategy to elicit broadly neutralizing antibodies (bnAbs). BnAbs are extremely valuable because they neutralize genetically distant viral strains at the same time. To overcome its inherently low affinity to germline B cells, the aim of the study was to present GT-Env via different immobilization strategies densely arrayed on the surface of nanoparticles. We engineered a prefusion-stabilized GT-Env trimer with affinity to VRC01 germline B cells using a bioinformatics-supported design approach. Distinct glycan modifications and amino acid substitutions yielded a GT-Env trimer which bound to the receptor with a KD of 11.5 µM. Silica nanoparticles with 200 nm diameter (SiNPs) were used for the multivalent display of the novel GT-Env with a 15 nm mean centre-to-centre spacing either by site-specific, covalent conjugation or at random, non-specific adsorption. Oriented, covalent GT-Env conjugation revealed better binding of structure dependent bnAbs as compared to non-specifically adsorbed GT-Env. In addition, GT-Env covalently attached activated a B cell line expressing the germline VRC01 receptor at an EC50 value in the nanomolar range (4 nM), while soluble GT-Env required 1,000-fold higher concentrations to induce signalling. The significantly lower GT-Env concentration was likely required due to avidity effects, which were in the picomolar range. Thus, low affinity antigens may particularly benefit from a particulate and multivalent delivery. In future, SiNPs are ideal to be modified in a modular design with various GT-Env variants that target different stages of germline and bnAb precursor B cells.