Figure 1.

Replicon constructs and replicon interaction with chitosan. (a) Gene arrangement of the classical swine fever virus (CSFV) genome parent for the replicon RNA (RepRNA) constructs (CSFV parent), showing the E^rns gene deletion for the ΔE^rns replicon. Insertion of the influenza virus hemagglutinin (HA) and nucleoprotein (NP) genes at the 3′ end of the N^pro gene are shown as HA replicon and NP replicon (the luciferase RepRNA has the luciferase gene inserted at the same position). Insertion of the influenza virus HA at the 3′ end of the C gene is shown as C-HA-C replicon. The inserted EMCV IRES ensured reinitiation of translation of the RepRNA after the inserted influenza virus genes. (b) Encapsulation efficiency for RepRNA interacting with chitosan during the formation of the chitosan cores of the nanoparticles. The RepRNA was labeled with FITC. Chitosan-based nanoparticles (NGA) are detected by their side scatter (or forward scatter, data not shown) profiles in the Flow Cytometer; RepRNA is detected in the FL-1 channel. The signal level with diluent alone (MQ-water) or NGA alone set the gating for positive FL-1 signals. Different concentrations of RepRNA were associated with the NGA (from 2 to 16 μg RepRNA in 17 μl 0.1% TPP plus 250 μl chitosan, made up to 1 ml prior to analysis). (c) Physical characteristics of the NGA, alone or carrying oligoRNA or RepRNA cargoes, measured in water (the standard diluent for NGA production) or in Dulbecco's modified Eagle's medium (DMEM) (the medium in which the NGA were employed for interaction with dendritic cells (DCs)). Measurement of the hydrodynamic diameter (Z-average size, d_HZ), polydispersity index (PDI) and surface charge (ζ-potential) was by dynamic light scattering at 25 °C with a scattering angle of 173°. (d) Capacity of chitosan to protect fluorochrome-labeled RNA probes from RNAse-dependent release of the fluorochrome, using the RNaseAlert Kit. Interaction of chitosan with the RNA in either water or Tris buffer was compared with RNA alone (in Tris buffer is shown; in water gave the same results) and RNA with TPP in water or Tris buffer. (e) Influence of chitosan on protecting the integrity of a Dy-781 labeled 50-mer RNA probe against RNase digestion, using the procedure described by Python et al.⁴⁹ TPP, tripolyphosphate.