In Vitro and In Vivo Stability of P884T, a Mutation that Relocalizes Dengue Virus 2 Non-structural Protein 5

Abstract

Dengue virus (DENV) non-structural protein 5 (NS5) is critical for viral RNA synthesis within endoplasmic reticulum (ER)-derived replication complexes in the cytoplasm; however a proportion of NS5 is known to be localized to the nucleus of infected cells. The importance of nuclear DENV NS5 on viral replication and pathogenesis is still unclear. We recently discovered a nuclear localization signal (NLS) residing in the Cterminal 18 amino acid (Cter 18 ) region of DENV NS5 and that a single NS5 P884T amino acid substitution adjacent to the NLS is sufficient to relocalize a significant proportion of DENV2 NS5 from the nucleus to the cytoplasm of infected cells. Here, in vitro studies show that the DENV2 NS5 P884T mutant replicates similarly to the parental wild-type infectious clone-derived virus while inducing a greater type I interferon and inflammatory cytokine response, in a manner independent of NS5's ability to degrade STAT2 or regulate SAT1 splicing. In both AG129 mouse and Aedes aegypti mosquito infection models, the P884T virus exhibits lower levels of viral replication only at early timepoints. Intriguingly, there appears to be a tendency for selection pressure to revert to the wild-type proline in P884T-infected Ae. aegypti, in agreement with the high conservation of the proline at this position of NS5 in DENV2, 3, and 4. These results suggest that the predominant nuclear localization of DENV NS5, while not required for viral RNA replication, may play a role in pathogenesis and modulation of the host immune response and contribute to viral fitness in the mosquito host.

Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization

“…We generated viruses containing the analogous mutation in DENV3 (P884T) and DENV4 (P885T) and the reverse T883P mutation in DENV1 and infected Huh7 cells for immunofluorescence assay (IFA) to visualize their NS5 subcellular localization. DENV2 wild-type (WT) and P884T mutant infectious clone-derived viruses that had previously been generated were used as controls …”

“…Despite this vital cytoplasmic role, flavivirus NS5 has been observed within the nucleus of infected cells, most notably in the case of DENV, ZIKV, and YFV, − but less so for WNV, JEV, Usutu virus, and Duck Tembusu virus. − It has been proposed that nuclear NS5 is important for modulation of the host immune response; previous studies have focused on the association between nuclear NS5 and induction of type I IFN response ,, as well as interactions between DENV2 NS5 and the spliceosome. , However, it remains an open question as to whether nuclear NS5 plays a critical role in DENV pathogenesis. A functional, virological approach to answering this question is to generate mutant viruses with relocalized NS5 for comparison with the wild-type DENV, in order to assess the impact of nuclear NS5 on defined pathogenesis markers.…”

Functional analysis of flavivirus replicase by deep mutational scanning of dengue NS5

“…Multi-sequence alignment of flavivirus NS5: Multi-sequence alignment (MSA) of NS5 was done with MAFFT version 7 (Katoh and Standley, 2013) using 95 non-segmented flavivirus representative NS5 sequences commissioned by the International Committee on Taxonomy of Viruses (ICTV, Supplementary Figure 4A). The MSA was displayed using MView (https://www.ebi.ac.uk/Tools/msa/mview/, Brown et al 1998) and WebLogo plot (https://weblogo.berkeley.edu/logo.cgi, Crooks et al 2004) (Supplementary Figure 4C). Phylogenetic tree based on the NS5 sequences (Supplementary Figure 4B) were constructed using the following setting on MAFFT web server: neighbor-joining algorithm, conserved sites (758 AAs), substitution model = JTT, ignore heterogeneity among sites, and bootstrap on with 100 number of resampling.…”

Let’s Get Physical: Flavivirus-Host Protein–Protein Interactions in Replication and Pathogenesis

“…For example, independent studies of ZIKV, WNV, and DENV NS5 all point to overall inhibition of immune gene expression ( López-Denman et al, 2021 ; Petit et al, 2021 ; Zhao et al, 2021 ). On the other hand, studies involving infection have not revealed in vitro or in vivo phenotypes for DENV mutants that reduce NS5 nuclear localization ( Kumar et al, 2016 ; Tay et al, 2016 ; Cheng et al, 2021 ), resulting in skepticism regarding the biological significance of NS5 nuclear localization. However, for studies involving DENV serotype 2, it should be noted that there are two distinct nuclear localization signals (NLSs) that contribute to nuclear localization.…”

“…The multiple mechanisms by which flaviviruses antagonize DNA sensing suggest that this is an important Flavivirus NS5 proteins also antagonize innate immunity upstream of IFN production and signaling, primarily through interactions with host gene expression machinery in the nucleus. The extent of NS5 localization to the nucleus varies depending on flavivirus species, yet some nuclear localization and nuclear/cytoplasmic shuttling appears to occur for nearly all NS5s, with the exception of duck Tembusu virus (Hannemann et al, 2013;Zhao et al, 2015;Grant et al, 2016;Kumar et al, 2016;Tay et al, 2016;Duan et al, 2019;Cheng et al, 2021;Petit et al, 2021). NS5 protein interactions with host gene expression machinery have been noted through several unbiased screens, including mass spectrometry and yeasttwo-hybrid screens (Khadka et al, 2011;Le Breton et al, 2011;De Maio et al, 2016;Coyaud et al, 2018;Scaturro et al, 2018;Shah et al, 2018;Li et al, 2019).…”