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.

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

“…Second, we created a conditionally replication-defective reporter by incorporating a large, in-frame deletion in the essential NS1 gene, which can be supplied in trans (23,57). The NS1 glycoprotein, which is expressed within the secretory pathway, contributes to the cytosolic process of RNA replication via interaction with the polytopic NS4A and NS4B membrane proteins, likely within the NS4A-2K-NS4B polyprotein intermediate (58)(59)(60). NS1 also has distinct membrane alteration properties (59,61), which likely contribute to replication complex assembly.…”

“…The NS1 glycoprotein, which is expressed within the secretory pathway, contributes to the cytosolic process of RNA replication via interaction with the polytopic NS4A and NS4B membrane proteins, likely within the NS4A-2K-NS4B polyprotein intermediate (58)(59)(60). NS1 also has distinct membrane alteration properties (59,61), which likely contribute to replication complex assembly. In the absence of NS1 expression, flavivirus infections are halted prior to replication complex formation and the initial round of RNA synthesis (23,59,62,63).…”

“…NS1 also has distinct membrane alteration properties (59,61), which likely contribute to replication complex assembly. In the absence of NS1 expression, flavivirus infections are halted prior to replication complex formation and the initial round of RNA synthesis (23,59,62,63). Consistent with this, robust Nluc expression by YFV-17D/Nluc was sensitive to a YFV-specific RNA replication inhibitor, BDAA, while the modest Nluc expression by YFVΔSK/Nluc was not.…”

Zika NS1–induced ER remodeling is essential for viral replication

“…In fact, we found that NS1 is indispensable for the replication compartment biogenesis; NS1 alone is sufficient to induce ER remodeling with architecture similar to replication compartments. Previous studies unraveled genetic and/or physical interaction of NS1 with NS4A and NS4B; however, it was recently reported that this interaction had no relationship with the formation of the replication compartment (Lindenbach and Rice, 1999; Płaszczyca et al, 2019; Youn et al, 2012). Thus, it is possible that ER lumen–localized NS1 regulates flavivirus replication via remodeling the ER structure or organizing the assembly of replication complexes.…”

Meeting report: 32nd International Conference on Antiviral Research

“…He noted that a compound currently under development at Janssen Pharmaceuticals targets the NS4B of all four dengue virus serotypes, while some other candidate molecules are not active against all serotypes. A recent paper from the Bartenschlager group validates the dengue NS1 protein as a "Swiss army knife" that could be an ideal antiviral target', especially given its interaction with precursor versions of NS4B (Plaszczyca et al, 2019). Opportunities for targeting dengue and Zika virus with nucleoside-analogue inhibitors such as remdesivir (Gilead) were also suggested, based on extensive structural and functional similarities of the NS5 protein with the replication mechanisms of other RNA viruses.…”