Affiliation:
1. School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
2. Molecular Biology Institute of Barcelona (CSIC), Barcelona Science Park (PCB), Barcelona, Spain
Abstract
ABSTRACT
The
Picornaviridae
is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in
trans
(i.e., via expression from a separate RNA molecule), while others are required in
cis
(i.e., expressed from the template RNA molecule).
In vitro
studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic
cis
-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this
cis
-acting role of 3D is distinct from the catalytic activity, which is predominantly
trans
acting. Immunofluorescence studies suggest that both
cis
- and
trans
-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in
cis
with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.
IMPORTANCE
Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in
trans
) while others must originate from the template (i.e., in
cis
). Here, we present an analysis of
cis
and
trans
activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel
cis
-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.
Funder
Biotechnology and Biological Sciences Research Council
Ministerio de Economía y Competitividad
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Cited by
18 articles.
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