Membrane Retention of West Nile Virus NS5 Depends on NS1 or NS3 for Enzymatic Activity

Abstract

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Keywords: NS1; NS3; NS5; West Nile virus; confocal microscopy; endoplasmic reticulum; flavivirus; replication organelles; sucrose density gradient.

Figure 1

WNV NS5 is localized to the endoplasmic reticulum membranes during infection. HEK293T cells were infected with WNV_NY99 at a MOI of 1 and harvested after 48 h. The cell fractions were separated using sucrose gradient ultracentrifugation and subjected to SDS-PAGE before immunoblotting. Viral proteins were detected using anti-WNV NS5, NS3, and NS2B antibodies. The lighter soluble fractions and heavier membrane fractions were visualized using antibodies against tubulin and calnexin, respectively.

Figure 2

WNV is localized to the cytoplasm and nucleus in the transfected cells. (A) (a–c) NS5-GFP-transfected or (d) NS5-GFP and IKKε-FLAG co-transfected HEK293T cells were fixed at 24 h post-transfection and immunostained with antibodies against (a) calnexin (red), (b) giantin (red), (c) tubulin (red), or (d) IKKε (red). Antibodies that detect the host markers and the viral protein’s GFP tag (green) are listed in the top right corner of each panel. Nuclear DNA (blue) was stained with 4,6-diamidino-2-phenylindole (DAPI). The confocal microscopy images were of optical slice thickness ~1 μm. Scale bar, 10 μM. The main image depicts the merged image from the three separate channels shown on the side. The white arrow indicates colocalization between the green and red channels. (B) The Pearson’s correlation coefficient (PCC) analysis of the colocalization between NS5 and cellular markers. PCC values at 0.5 (dotted line) indicate a high level of colocalization. Letters in parenthesis correspond to its representative confocal image in Figure 2A. Error bars indicate the mean ± standard error of the mean (SEM); n = 7–10 cells per group. (C) HEK293T cells were transfected with NS5-GFP for 48 h and subjected to subcellular fractionation followed by sucrose gradient centrifugation. The cell fractions were analyzed by Western blot using rabbit anti-GFP antibody to detect NS5. The membrane and soluble fractions were detected using antibodies against calnexin and tubulin, respectively.

Figure 3

WNV NS5 is associated with the RER when it is tethered to NS4B. (A) The NS4B-NS5-GFP (green) and sec61β-RFP (red) co-transfected HEK293T cells were fixed 24 h after transfection and immunolabeled with anti-JEV NS4B (blue). Optical slice thickness ~1 μm. Scale bar, 10 μM. The white arrow indicates colocalization between all three channels. The image is representative of three independent transfection experiments. (B) Pearson’s correlation coefficient between NS4B-NS5 and sec61β (RER marker). PCC values greater than 0.5 (dotted line) indicate a high level of colocalization. Error bars indicate mean ± SEM; n = 10 cells. (C) The cells transiently expressing NS4B-NS5-GFP were harvested after 48 h. The cell fractions separated using SDGU were immunoblotted with antibodies against GFP, calnexin, and tubulin.

Figure 4

Processing of WNV NS5 from the NS4B-NS5 polyprotein does not influence its RER localization. (A) The schematic representation of the V5/His (red box) or GFP-tagged (green box) WNV NS constructs used in this assay. Constructs are drawn to scale according to the number of amino acid residues. (B) HEK293T cells were transfected with various WNV NS constructs (listed at the top of the panel), and lysates were harvested 48 h post-transfection. The cleavage of NS4B-NS5-GFP by the viral protease (NS3-V5/His with NS2B-V5/His provided in trans) in lane 5 was assessed by Western blot. (C) HEK293T cells were transfected with the NS4B-NS5-GFP (green), NS2B-V5, NS3-V5, and sec61β-RFP (red) plasmids, and the quadruple-transfected cells were processed for immunofluorescence 24 h after transfection. Optical slice thickness ~1 μm. Scale bar, 10 μM. White arrows indicate colocalization between sec61β-RFP and NS4B (blue). (D) Colocalization analysis between the cleaved NS5 protein, sec61β, and NS4B. Error bars indicate mean ± SEM; n = 8 cells. PCC values above 0.5 (dotted line) indicate higher levels of colocalization. (E) Cell fractions from cells expressing NS4B-NS5-GFP, NS2B-V5, NS3-V5, and sec61β-RFP 48 h post-transfection were analyzed by Western blot. The cleaved NS5 was detected using an anti-GFP antibody and the NS2B and NS3 proteins were visualized using anti-V5/His antibodies. The membrane and soluble fractions were distinguished using anti-calnexin and anti-tubulin antibodies, respectively.

Figure 5

WNV NS5 predominantly localizes in the cytoplasm and partially localized in the membrane fractions when NS3 is present. (A) HEK293T cells were co-transfected with (a–c) the NS5-GFP (green) and NS3-V5/His (blue) plasmids or (d) triple-transfected with the NS5-GFP, NS3-V5/His and IKKε-FLAG plasmids. The cells were processed for immunofluorescence 24 h after transfection using antibodies against (a) calnexin (red), (b) giantin (red), (c) tubulin (red), or (d) IKKε (red). White arrows depict colocalization between (a–c) the green and blue channels or (d) all three channels. Scale bar, 10 μM. (B,C) Pearson’s correlation coefficient to measure colocalization was determined for each indicated pair of proteins. PCC values at 0.5 (dotted line) indicate a high level of colocalization. Parenthesized letters link each PCC value to its corresponding image in Figure 5A. The PCC for NS5 and NS3 was calculated using the co-transfected (a–c) and triple-transfected (d) cells. Error bars indicate mean ± SEM; n = 7–10 cells per group. (D) Subcellular fractions from cells expressing NS5-GFP with NS3-V5/His provided in trans 48 h post-transfection were analyzed using antibodies against the fused tag (GFP or V5/His) to detect the viral proteins. Calnexin and tubulin indicate the membrane and soluble fractions, respectively.

Figure 6

WNV NS5 is distributed throughout the cytoplasm and nucleus, and is slightly enriched in the membrane fractions when NS1 is present. (A) HEK293T cells were co-transfected with (a–c) the NS5-GFP (green) and NS1-V5/His (blue) plasmids or (d) triple-transfected with the NS5-GFP, NS1-V5/His and IKKε-FLAG plasmids. The cells fixed 24 h after transfection were immunostained with antibodies against (a) calnexin (red), (b) giantin (red), (c) tubulin (red), and (d) IKKε (red). White arrows depict colocalization between (a,b) the red and blue channels or (d) between the red and green channels. Scale bar, 10 μM. Pearson’s correlation coefficient was calculated for (B) NS5 and the host markers in the presence of NS1 and for (C) NS5 and NS1. PCC values greater than 0.5 (dotted line) indicate high levels of colocalization. Letters in parenthesis correlate each PCC value to its representative image in (A). Error bars indicate mean ± SEM; n = 7–10 cells per group. Error bars indicate mean ± SEM; n = 10 cells. (D) The cellular fractions from HEK293T cells co-expressing NS5-GFP with NS1-V5/His were analyzed using antibodies against the fused tag (GFP or V5/His) to detect the viral proteins, and against calnexin or tubulin to visualize the membrane and soluble fractions, respectively.