Ledbetter, A. | bioRxiv Science

bioRxiv2026

Structural Basis for Dimeric Genome Selection by HIV-1

HIV-1 packages a dimeric RNA genome into assembling virions, a requirement for replication. To understand the mechanism of dimer selection, we conducted cryogenic electron microscopy (cryoEM) studies of nascent assemblies formed between HIV-1 Gag protein constructs and genomic RNA elements that promote dimerization and packaging. CryoEM maps revealed structural architectures in which the helical dimer initiation signal (DIS) of the RNA packaging signal bridges two 6-helix bundles (6HBs) that protrude from adjacent Gag hexamers. Conserved guanosines that sandwich the DIS are located beneath the 6HBs and poised to interact with clusters of nucleocapsid domains. Mutation of these guanosines severely impairs RNA packaging into virus-like particles. Our findings suggest that DIS functions as a molecular ruler, organizing Gag hexamers into structures optimized for Gag lattice expansion.

molecular biology↗

bioRxiv2025

Engineering gene expression dynamics via self-amplifying RNA with drug-responsive non-structural proteins

The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug- regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.

bioengineering↗