Zika virus NS3 drives the assembly of a viroplasm-like structure

Abstract

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused an epidemic in 2015-2016 in the Americas and raised serious global health concerns due to its association with congenital brain developmental defects in infected pregnancies. Upon infection, ZIKV assembles virus particles in a virus-generated toroidal compartment next to the nucleus called the replication factory, or viroplasm, which forms by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control viroplasm assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3) is sufficient to drive the assembly of a viroplasm-like structure (VLS) in human cells. NS3 encodes a dual-function protease and RNA helicase. The VLS is similar to the ZIKV viroplasm in its assembly near centrosomes at the nuclear periphery, its deformation of the nuclear membrane, its recruitment of ER, Golgi, and dsRNA, and its association with microtubules at its surface. While sufficient to generate a VLS, NS3 is less efficient in several aspects compared to viroplasm formation upon ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal VLS assembly and dsRNA recruitment. Overall, this work advances our understanding of the mechanism of viroplasm assembly by ZIKV and likely will extend to other flaviviruses.

Figure 1.. ZIKV NS3 drives assembly of a viroplasm like structure (VLS) which precedes ER recruitment.

(A) Schematic of the ZIKV polyprotein and its topology within the ER membrane. Created with Biorender.com. Immunofluorescent (IF) staining of (B) mock and ZIKV-infected cells 24h post-infection (p.i) (viroplasm marked by ZIKV envelope protein; green) and (B′) mock and FLAG-NS3 transfected for 48h (NS3-driven VLS marked by FLAG; green) SNB19 cells. ER is marked with calnexin (red). (C) Morphology of the NS3-driven VLS. ER recruitment (D) to the viroplasm, marked with NS3, 16h (upper panel), 20h (middle panel), 24h (bottom panel) p.i and to the VLS (D′) 24h after transfection (upper panel) and 48h (bottom panel). ER is marked with KDEL (red). DAPI was used to stain the nucleus (blue) for IF staining in all figures. Scale bars: 10 μm.

Figure 2.. NS3-driven VLS forms in association with the centrosome and recruits the Golgi apparatus.

IF staining for the centrosome protein CEP192 (red) in (A) mock and ZIKV (MR766) 24h post-infection and (A′) mock and FLAG-NS3 transfected for 48h in SNB19 cells. ZIKV-induced viroplasm (envelope protein, green) and NS3-driven VLS (FLAG, green). Illustration (B) and bar graph (mean ± standard deviation) shows different localization of centrosome in NS3-driven VLS. IF staining of the Golgi protein GM130 (red) in (D) mock and ZIKV infection for 24h and (D′) mock and FLAG-NS3 transfected for 48h in SNB-19 cells. The Golgi at the core is indicated by yellow arrowheads and the Golgi surrounding the viroplasm by white arrowheads. Scale bars: 10 μm.

Figure 3.. The NS3-driven VLS recruits dsRNA

IF staining of dsRNA (rJ2 antibody, white) (A) in mock and ZIKV (MR766) 24h post-infection. ZIKV-induced viroplasm is marked by ZIKV envelope protein; red. (B) FLAG-NS3 transfection (top panel) and fluorescein-labeled (Flsn) dsRNA transfection (middle panel) and co-trasnfection of Flsn dsRNA with FLAG-NS3 (bottom panel) for 48h in SNB19 cells. dsRNA recruitment is indicated by the yellow arrowheads. NS3-driven VLS is marked with FLAG (red). Scale bars: 10 μm.

Figure 4.. Microtubules are reorganized at the NS3-driven VLS.

IF staining of microtubules (MTs) marked by α-tubulin (red) in (A) mock and ZIKV 24h post-infection and (B) mock and FLAG-NS3 transfected for 48h in SNB19 cells. ZIKV-induced viroplasm (green) and NS3-driven VLS (green) are marked with Envelope and FLAG respectively. Yellow arrowheads indicate MTs arrangement around the viroplasm and NS3-driven VLS. Scale bars: 10 μm.

Figure 5.. VLS formation by NS3 requires the ATPase domain

(A) NS3 structure denoting protease and helicase domains and positions of introduced mutations. (B) Western blot of FLAG-tagged wild-type and mutant NS3 proteins. (C) IF staining of FLAG-NS3 and mutated NS3 (S135A, K210N, D290N and R461Q) transfected for 48h in SNB19 cells. NS3-driven VLS is marked with FLAG (green). (D) Circularity of VLS with different mutations. Different colors indicate different experiments (n=4). *P < 0.05, ** P < 0.001, and ***P < 0.0001 are considered statistically significant differences from the control (FLAG-NS3). DAPI stains the nucleus (magenta). Scale bars: 10 μm.