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. 2001 Feb;75(4):1808-15.
doi: 10.1128/JVI.75.4.1808-1815.2001.

Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region

E V Ravkov  1 , R W Compans
Affiliations

Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region

E V Ravkov et al. J Virol. 2001 Feb.

Abstract

Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with alpha-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of the Bunyaviridae family.

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Figures

FIG. 1
FIG. 1
Intracellular localization of NBCCV. BHK21 cells were seeded onto coverslips in a 12-well plate, grown to 60% confluency, and transfected with pcNBCCV. The cells were fixed at 24 h posttransfection, and the NBCCV antigen was identified with IFA by using anti-N MAb (A). The cells were costained with phalloidin to show cellular morphology (B). The NBCCV antigen staining is concentrated in the perinuclear region of each transfected cell. A few cells also exhibit a filamentous pattern of the antigen. Panels C and D show BHK21 cells coexpressing the NBCCV and NLACV proteins. The cells were transfected with the pcNBCCV and pcNLACV plasmids and examined by double immunofluorescence staining at 24 h posttransfection. Panel C shows staining with anti-BCCV N MAb, and panel D shows staining with anti-NLACV rabbit polyclonal antibody. While the NLACV antigen is localized diffusely in the cytoplasm, the NBCCV antigen is found in the perinuclear region in the same cells.
FIG. 2
FIG. 2
NBCCV is localized in the Golgi region. Panels A and B show BHK21 cells that were dispersedly seeded and transfected with the pcNBCCV plasmid. The cells were examined by double immunofluorescence staining at 24 h posttransfection. Panel A represents staining with anti-N MAb, and panel B shows staining with anti-α-mannosidase II polyclonal antibody. Panels C and D show cells that were separately transfected with the plasmid pGFP/NBCCV (C), driving expression of NBCCV fused with GFP. Panel D represents cells transfected with pEGFP-c3, driving expression of GFP alone. The cells were examined at 24 h posttransfection by direct GFP fluorescence.
FIG. 3
FIG. 3
Identification of the NBCCV perinuclear localization sequence. Panel A shows a diagram of the NBCCV C- and N-terminally truncated mutants. The numbers indicate the position of ATG and TGA codons in the constructs relative to the amino acid sequence of the NBCCV protein. Panel B shows IFA of BHK21 cells expressing the NBCCV truncated proteins. The cells were fixed in 4% paraformaldehyde at 24 h posttransfection, permeabilized, and stained with either anti-N MAb (the upper row) or anti-GFP MAb (the lower row).
FIG. 4
FIG. 4
NBCCV is associated with microsomal membranes. BHK21 cells were harvested at 24 h posttransfection, disrupted in a homogenizer, and subjected to differential centrifugation to obtain microsomal (M) and cytosolic (C) fractions. The proteins of both fractions were separated by SDS-PAGE and analyzed by Western blotting with a specific antibody. The first row shows detection of β-COP (expressed endogenously), GFP, NBCCV, and GFP-NBCCV with anti-β-COP, anti-GFP, and anti-N MAbs, respectively. The second row represents subcellular fractionation of BHK21 cells expressing the C-terminally truncated proteins, which were detected with anti-N MAb. The third row shows detection of GFP-N-terminally truncated mutants with anti-GFP MAb.
FIG. 5
FIG. 5
Membrane association properties of NBCCV. NBCCV-microsomes that were either untreated (the first gel from top) or treated with 1 M NaCl (second gel), 4 M urea (third gel), or 15 mM sodium carbonate (pH 11) (fourth gel) were analyzed in sucrose density gradients. The fractions (∼450 μl each) were collected from the bottom of the gradient, precipitated with either ethanol or acetone, and examined by Western blotting with anti-N MAb. Lane numbers correspond to fractions collected from the gradients.
FIG. 6
FIG. 6
NPUUV and NSEOV are localized to the perinuclear region. The intracellular localization of these proteins was examined by IFA. BHK21 cells were grown on coverslips and transfected either with pcNPUUV or pcNSEOV plasmids. At 24 h posttransfection, the cells were examined by IFA with anti-N MAb as the first antibody. Panel A shows expression of NPUUV, and panel B shows intracellular localization of NSEOV.
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