Knots and Crosses: The roles of RNA structure and the 3B protein in foot-and-mouth disease virus replication

Foot-and-mouth disease virus (FMDV) is a single stranded RNA virus in the picornavirus
family. It is the causative agent of foot-and-mouth disease, globally the
most important a�iction of cloven hoofed animals. The FMDV genome has several
features that are not found amongst other viruses within the Picornaviridae. These
include a large 50 untranslated region (UTR), almost twice the length of that found
in enteroviruses, containing highly structured RNA elements unique to FMDV, such
as the S-fragment and several tandemly repeated pseudoknots. Unique aspects are
also found within the coding region, where FMDV is the only picornavirus reported
to contain multiple copies of the 3B gene. The reasons behind possession of these
unusual deviations from the dogma of the picornaviral genome is so far unknown
and therefore poses an attractive target for further research.
The S-fragment is a predicted 360 nucleotide stem-loop at the 50 end of the FMDV
genome. The better studied poliovirus (PV) has a well characterised 80 nucleotide
clover leaf structure in the equivalent position which is used as a model for potential
S-fragment function. In close proximity to the S-fragment are a series of tandemly
repeated pseudoknots, these pseudoknots are maintained across all sequenced isolates
of FMDV, however, the number of pseudoknots varies between two and four
depending on the specifc isolate. As with the S-fragment, no function is yet assigned
to these pseudoknots and the reason behind the variation in number remains
unknown. Here, biochemical probing of the S-fragment and pseudoknots has been
used to confirm the secondary structures of these elements and helped inform mutagenesis
experiments to disrupt conformations. Sub-genomic FMDV replicons are
used in tandem with infectious virus to help dissect the roles of these elements in
distinct areas of the viral lifecycle.
Alongside RNA structure in the 50 UTR, this thesis also investigates the nonstructural
P3 region and how the 3B protein plays a key role in correct polyprotein
processing. The complex dynamics of picornavirus polyprotein processing generates
intermediate products which expand the repertoire of functions encoded in the
genome. Dissecting these processes is therefore key for understanding the details
of virus replication. As the polyprotein of FMDV uniquely includes three tandem
functional copies of the 3B primer peptide, the manipulation of P3 cleavage sites is
used here to further uncover the roles of the 3B protein in replication. The importance
of the C-terminal region of 3B3 for correct polyprotein processing is described
as well as the capabilities for 3B proteins to accept C-terminal fusions, providing a
novel set of molecular tools and approaches to further analyse the complexities of
picornavirus genome replication in the future.