Genome-wide mutagenesis of dengue virus reveals plasticity of the NS1 protein and enables generation of infectious tagged reporter viruses

ABSTRACT

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and sub-tropical 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 development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and employed next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that regions within capsid, NS1 and the 3’ UTR were 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. Additionally, 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 genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.

IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an ‘APEX2’ -tagged virus that we employed in high resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time this analysis revealed the localization of NS1 within viral replication factories known as ‘vesicle packets’ (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together this genetic profile of DENV may be further refined and exploited in identification of antiviral targets and generation of reporter virus tools.