Abstract (eng)
Viral genome transfer through cellular membranes is a crucial process in the course of early infection events of non-enveloped viruses. It is the aim of this work to contribute to a better understanding of this mechanism taking human Rhinoviruses as typical representative of this virus type. They contain an RNA genome of about 7 kb in an icosahedral protein capsid, which needs to be transferred through the endosomal membrane to infect the host cell. This work aimed at detecting in-vitro the essential viral and cellular elements for proper genome transfer.
Instead of using natural cells with native receptors, the liposome-based model used a nickel chelating lipid for attaching His6-tagged receptor constructs, derived from the very low-density lipoprotein receptor (VLDLR). Upon specific binding of HRV2, the assembly was primed for infection, and thus called “lipofectosome”. The suitability of this model was confirmed by capillary electrophoresis (CE) and transmission electron microscopy (TEM). Finally, proper genome transfer could be demonstrated by a reverse transcription (RT) PCR assay.
Parameters for assembly were optimized by CE with laser-induced fluorescence (LIF) detection and TEM imaging. The latter technique enabled screening of samples kept in moderately acidic environment to identify conditions for entire RNA release.
To confirm the ingress of released RNA into the liposomal compartment, an RT protocol was adapted to allow for its application within lipofectosomes. It was shown that upon triggering infection, lipofectosomes transferred the RNA from within the virion into the interior of the vesicle; their viral RNA was detected via RT and PCR. Since liposomes served here as leak-tight compartment to carry out RT, they can be considered as nano-reaction containers.
In conclusion, a system to monitor in-vitro the RNA transfer of non-enveloped viruses was invented. This work shows that even complex infection pathways can be resolved to discrete and plain processes, such as the genome transfer.