A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle

Abstract

Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the β-ladder domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “ Wing” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a trans-complementation assay, we show that both cis and trans acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.

Author summary

Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human pathogens. The only licensed vaccine has limited efficacy and an antiviral therapy is not available. The multifunctional non-structural protein 1 (NS1) of DENV is secreted from infected cells, counteracts antiviral immune response and contributes to the pathogenesis of DENV infection. In addition, NS1 is essential for the viral replication cycle but the underlying mechanism is unknown. Here we determined the viral interactome of NS1 and identified a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with NS4A and NS4B. This interaction is required for RNA replication. Additionally, we identified a domain in NS1 important for efficient secretion of this protein. Finally, we demonstrate that NS1 is required for the biogenesis of the membranous DENV replication organelle. This function does not require RNA replication and is independent from NS1 interaction with NS4A-2K-4B. Our results provide new insights into the role of NS1 in DENV RNA replication and establish a genetic map of residues in NS1 required for the diverse functions of this protein. These results should aid in the design of antiviral strategies targeting NS1, with the aim to suppress viral replication as well as severe disease manifestations.