Functional Analysis of the Dengue Virus Genome Using an Insertional Mutagenesis Screen.

In the last few decades, dengue virus, an arbovirus, has spread to over 120 countries. Although a vaccine has been approved in some countries, limitations on its effectiveness and a lack of effective antiviral treatments reinforce the need for additional research. The functions of several viral nonstructural proteins are essentially unknown. To better understand the functions of these proteins and thus dengue virus pathogenesis, we embarked on a genomewide transposon mutagenesis screen with next-generation sequencing to determine sites in the viral genome that tolerate 15-nucleotide insertions. Using this approach, we generated support for several published predicted transmembrane and enzymatic domains. Next, we created 7 mutants containing the 15-nucleotide insertion from the original selection and found 6 of them were capable of replication in both mammalian and mosquito tissue culture cells. Interestingly, one mutation had a significant impairment of viral assembly, and this mutation may lead to a better understanding of viral assembly and release. In addition, we created a fully infectious virus expressing a functionally tagged NS4B protein, which will provide a much-needed tool to elucidate the role of NS4B in viral pathogenesis.