TY - JOUR
T1 - Genome-wide mutagenesis of dengue virus reveals plasticity of the NS1 protein and enables generation of infectious tagged reporter viruses.
AU - Eyre, Nicholas S
AU - Johnson, Stephen M
AU - Eltahla, Auda A.
AU - Aloi, Maria
AU - Aloia, Amanda L
AU - McDevitt, Christopher A.
AU - Bull, Rowena A
AU - Beard, Michael R
PY - 2017/12/1
Y1 - 2017/12/1
N2 - Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3= untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genomewide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.
AB - Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3= untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genomewide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.
KW - Dengue virus
KW - NS1 Protein
KW - Virus
KW - Virus assembly
KW - Mutagenesis
KW - Virus replication
KW - Electron microscopy
KW - NS1
UR - http://purl.org/au-research/grants/NHMRC/1084706
UR - http://purl.org/au-research/grants/NHMRC/1130128
UR - http://purl.org/au-research/grants/NHMRC/1053206
UR - http://purl.org/au-research/grants/NHMRC/1027641
UR - http://www.scopus.com/inward/record.url?scp=85033776499&partnerID=8YFLogxK
U2 - 10.1128/JVI.01455-17
DO - 10.1128/JVI.01455-17
M3 - Article
VL - 91
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 23
M1 - e01455-17
ER -