Cis-acting RNA signals are required for
replication of positive-strand viruses such as the picornaviruses.
Although these generally have been mapped to the 5′
and/or 3′ termini of the viral genome, RNAs derived
from human rhinovirus type 14 are unable to replicate unless
they contain an internal cis-acting replication
element (cre) located within the genome segment
encoding the capsid proteins. Here, we show that the essential
cre sequence is 83–96 nt in length and located
between nt 2318–2413 of the genome. Using dicistronic
RNAs in which translation of the P1 and P2–P3 segments
of the polyprotein were functionally dissociated, we further
demonstrate that translation of the cre sequence
is not required for RNA replication. Thus, although it
is located within a protein-coding segment of the genome,
the cre functions as an RNA entity. Computer folds
suggested that cre sequences could form a stable
structure in either positive- or minus-strand RNA. However,
an analysis of mutant RNAs containing multiple covariant
and non-covariant nucleotide substitutions within these
putative structures demonstrated that only the predicted
positive-strand structure is essential for efficient RNA
replication. The absence of detectable minus-strand synthesis
from RNAs that lack the cre suggests that the
cre is required for initiation of minus-strand
RNA synthesis. Since a lethal 3′ noncoding region
mutation could be partially rescued by a compensating mutation
within the cre, the cre appears to participate
in a long-range RNA–RNA interaction required for
this process. These data provide novel insight into the
mechanisms of replication of a positive-strand RNA virus,
as they define the involvement of an internally located
RNA structure in the recognition of viral RNA by the viral
replicase complex. Since internally located RNA replication
signals have been shown to exist in several other positive-strand
RNA virus families, these observations are potentially
relevant to a wide array of related viruses.