A structural and functional analysis of opal stop codon read-through during Chikungunya virus replication

Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus
belonging to the alphavirus genus within the Togaviridae family. It is transmitted
by Aedes species mosquitoes. Whereas CHIKV infection in mosquitoes is
persistent and symptomless, the virus causes an acute onset of muscle and joint
pain, fever, nausea and headache in humans. Most infected individuals suffer
from persistent chronic joint and muscle pain after the initial infection is cleared.
CHIKV has been reported in over 50 countries worldwide, as yet there are no
specific antiviral agents or vaccines available.

The CHIKV genome contains two open reading frames. The 5′ of these encodes
a polyprotein that is cleaved to 4 non-structural proteins (nsP1-4). The third of
these (nsP3) is essential for virus replication but the exact function is unknown.
Some strains of CHIKV contain an in-frame opal stop codon near the C-terminus
of nsP3. Translational termination at this position would result in expression of a
C-terminally truncated nsP3 and a lack of nsP4 (the viral RNA dependant RNA
polymerase). Programmed read-through of this stop codon is necessary to
express all four nsPs.

The advantages conferred by possession of the opal stop codon are unknown.
To test this a panel of CHIKV subgenomic replicons and infectious clones with
either the opal, ochre or amber stop codon in place of the naturally occurring
arginine codon were generated. The presence of a stop codon reduced CHIKV
replication in mammalian cells but enhanced replication in mosquito cells.
SHAPE was used to determine the RNA structure surrounding the stop codon
and revealed a large 3′ stem-loop that may play a role in translational readthrough.
The data suggests that the stop codon has species-specific functionally,
in particular that the stop codon read-through signal may function more efficiently
in the invertebrate host.