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 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 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 the 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 used 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 the identification of antiviral targets and the generation of reporter virus tools.

Emergency Services of Viral RNAs: Repair and Remodeling

“…Various deletions and insertions in the 3= UTR as well as complete deletion of the poly(A) tail may not kill alphaviruses (457)(458)(459), flaviviruses (460)(461)(462), coronaviruses (463)(464)(465), and various plant viruses (466)(467)(468)(469). The repair of the damaged genomes may again involve different mechanisms, including RNA recombination, the use of the viral RdRP-or host-dependent polyadenylation activities, and perhaps some others.…”

In Vivo Dynamics of Reporter Flaviviridae Viruses

“…The five recombinant JEVs carrying the reporter genes within structural or nonstructural proteins exhibited comparable replication in vitro, and the recombinants carrying the tag in the nonstructural protein NS1 showed pathogenicity in mice. Recently, three different groups reported using whole-genome transposon mutagenesis screening of flaviviruses to identify tolerant loci for the insertion of foreign genes (31)(32)(33). Although the suitable loci identified in our present study overlapped those identified in these previous studies, our method has the additional advantage of narrowing down the number of viruses for screening.…”

Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone

“…This approach has been successfully applied to a number of positive-strand RNA viruses (Arumugaswami et al , 2008; Beitzel et al , 2010; Teterina et al , 2011; Thorne et al , 2012; Remenyi et al , 2014; Eyre et al , 2017; Fulton et al , 2017). By coupling the transposon mutagenesis approach to Next Generation Sequence, a map of sites in the viral genome that tolerate insertions can be determined with unprecedented resolution.…”