On the Genome Release Dynamics of Single-Stranded RNA Viruses
- Author(s): Sportsman, Richard
- Advisor(s): Gelbart, William M;
- Knobler, Charles M
- et al.
Measurements are described that begin to elucidate the mechanism by which some RNA viruses deliver their genetic information to their hosts. The work focuses on two closely related icosahedral viruses, Cowpea Chlorotic Mottle Virus and Brome Mosaic Virus. Each consists of a 28-nm diameter icosahedral protein shell, the capsid, containing an approximately 3000-nt long single-stranded RNA. We hypothesize that the RNA inside the capsid is delivered to the host’s ribosomes by a thermal fluctuation of an end of the RNA being made available in a process called co-translational delivery. In this process, an end of the RNA fluctuates out of an intact capsid, is captured by the ribosomal machinery and is pulled out of the capsid under force.
We conducted studies to characterize RNA fluctuations that occur through capsid pores by capturing an RNA end. To realize this capture we have utilized ribosomes and purified eukaryotic initiation factor complex eIF4F. This complex is known to initiate the cap-dependent translation process of mRNA by eukaryotic ribosomes. In other studies, the fluctuations of the RNA outside of the capsid were captured by utilizing biotinylated fluorescent RNA which were caught by streptavidin-coated surfaces or nanoparticles.
Additionally we obtained preliminary data using magnetic tweezers to measure the forces required to extract RNA from a capsid with a protruding RNA end. Rather than capture a fluctuation in this force measurement work, we designed a virus like particle construct dubbed a “cherry bomb”. We produced this construct by packaging, in vitro, an RNA where its 5’ terminus had been made rigid by hybridizing to it a length of complementary DNA that is sufficiently long, such that it cannot be accommodated within the virus capsid.
Finally, we investigated how RNA can be condensed at the single-molecule level using polyvalent cations and observe changes in how a condensed RNA can be packaged into virus like particles by viral capsid protein.
Our results hint at the inherent dynamics of the viral RNA genome and its capsid shell, and the complex processes involved in a virus interacting with its host, processes that have been difficult to measure using other structural biological or physical chemical techniques.