Severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain - PubMed (original) (raw)

Severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain

Shui-Mei Wang et al. J Biomed Sci. 2008 Nov.

Abstract

We replaced the HIV-1 nucleocapsid (NC) domain with different N-coding sequences to test SARS-CoV nucleocapsid (N) self-interaction capacity, and determined the capabilities of each chimera to direct virus-like particle (VLP) assembly. Analysis results indicate that the replacement of NC with the carboxyl-terminal half of the SARS-CoV N resulted in the production of wild type (wt)-level virus-like particles (VLPs) with the density of a wt HIV-1 particle. When co-expressed with SARS-CoV N, chimeras containing the N carboxyl-terminal half sequence efficiently packaged N. However, the same was not true for the chimera bearing the N amino-terminal half sequence, despite its production of substantial amounts of VLPs. According to further analysis, HIV-1 NC replacement with N residues 2-213, 215-421, or 234-421 resulted in efficient VLP production at levels comparable to that of wt HIV-1, but replacement with residues 215-359, 302-421, 2-168, or 2-86 failed to restore VLP production to wild-type levels. The results suggest that the domain conferring the ability to direct VLP assembly and release in SARS-CoV N is largely contained between residues 168 and 421.

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Figures

Fig. 1

Fig. 1

SARS-CoV nucleocapsid protein self-association. (a) Schematic representations of construct coding for recombinant SARS-CoV N proteins. CoN-myc directed by a CMV promoter encoded full-length SARS-CoV N tagged with Myc at carboxyl terminus. Boundaries of the RNA binding domain and self-association domain are indicated [41]. GST-CoN, GST-N1, and GST-N2 expressed GST fusion proteins with full-length, amino-terminal half, and carboxyl-terminal half of N fused to GST carboxyl terminus, respectively. (b, c) GST pull-down assay. 293T cells were co-transfected with 10 μg of CoN-myc and 10 μg of indicated GST fusion construct. Aliquots of cell lysates preceding (panel b) and following (panel c) GST pull-down were subjected to Western immunoblotting using anti-GST and anti-Myc antibodies as probes

Fig. 2

Fig. 2

Expression and assembly of chimeras containing SARS-CoV N coding sequences to serve as substitutes for the HIV-1 NC domain. (a) Schematic representations of wt HIV-1 Gag and mutant constructs. Shown are mature HIV-1 Gag protein domain matrix (MA), capsid (CA), nucleocapsid (NC), p6, and two spacer peptides (SP1 and SP2). ΔNC has ten HIV-1 NC residues remaining in the deleted region; ΔPC has NC almost deleted, with SP1 partially removed. delNC has the two methionine residues (bracketed) in the SP1-NC junction removed. Almost the entire (codons 2–421) or carboxyl-terminal half (215–421) of the SARS-CoV nucleocapsid protein (N) coding sequence was inserted into the deleted NC region, yielding the designated constructs. Arrows indicate SP1-NC and NC-SP2 junction sites. Remaining HIV-1 NC residues in deleted regions are underlined. Altered or foreign amino acid residues inserted in juncture area are italicized. Backbone was the protease-defective (PR−) expression vector HIVgptD25. (b, c) Expression and assembly of chimeric proteins. 293T cells were transfected with 20 μg plasmid DNA for each indicated construct. At 48 h post-transfection, cells and supernatant were collected for protein analysis as described in Materials and Methods. Cell samples corresponding to 4% of total cell lysates and supernatant samples corresponding to 50% of total recovered viral pellets were fractionated using 10% SDS-PAGE. HIV-1 Gag or chimeric proteins were probed an anti-p24CA monoclonal antibody or with a monoclonal antibody directed against N (panel b, upper panels). Positions of wt HIV-1 Gag, Gag-Pol and molecular size markers are indicated. p24CA-associated proteins from medium or cell samples were quantified by scanning mutant and wt band densities from immunoblots. Ratios of p24_gag_-associated protein levels in media to those in cells were determined for each construct and compared with wt release levels by dividing the release ratio for each mutant by the wt ratio in parallel experiments. Relative release factor (RF) values are indicated

Fig. 3

Fig. 3

Sucrose density gradient fractionation of wt and chimeric VLPs. (a) 293T cells were transfected with 20 μg plasmid DNA from wt, NC(CoN), or NC(N2). At 48 h post-transfection, culture supernatant was collected, filtered, and pelleted through 20% sucrose cushions. Viral pellets were resuspended in PBS, pooled, and centrifuged through a 20–60% sucrose gradient for 16 h. Ten equal-amount fractions were collected from top to bottom. Fraction densities were measured and wt and chimeric proteins analyzed by Western immunoblotting with an anti-p24CA antibody. (b) p24_gag_-associated wt and chimeric proteins were quantified via scanning densitometry. Band density units in each fraction were divided by total band density units of 10 fractions and multiplied by 100. Percentages of p24_gag_-associated protein in each fraction were plotted against the fraction’s sucrose density

Fig. 4

Fig. 4

Expression and assembly of chimeric protein containing a HIV-1 NC replacement consisting of SARS-CoV N coding sequences. (a) Schematic representation of chimeric constructs. Mature HIV-1 Gag protein domain and amino acid residues in SP1-NC-SP2 junction area are indicated. PCR-amplified fragments containing various portions of SARS-CoV N coding sequences were inserted into the deleted NC regions. Numbers denote codon positions at inserted SARS-CoV N protein sequence boundaries. All constructs were expressed in HIVgptD25 (a HIV-1 PR-defective expression vector). Expression and release of wt Gag and chimeric proteins. 293T cells were transfected with the indicated construct. ΔNC(wtZiP) and ΔNC(kZiP) contained HIV-1 NC replacements consisting of wild-type or a mutated version of a leucine zipper domain, respectively. At 48–72 h post-transfection, supernatant and cells were collected, prepared, and subjected to SDS-10% PAGE. HIV-1 Gag or chimeric proteins were detected by Western immunoblotting using monoclonal antibodies against SARS-CoV N or against HIV-1 p24CA. Positions of molecular size markers are indicated. Relative VLP release efficiency for each mutant was determined as described in the Fig. 2 caption; relative release factor values are indicated. (b) Expression and incorporation of SARS-CoV N protein into chimeric VLPs. 293T cells were co-transfected with 10 μg CoN-myc plus 10 μg pBlueScript SK or indicated construct. At 48–72 h post-transfection, cells and supernatant were collected and subjected to Western immunoblotting. SARS-CoV N protein was probed with an anti-Myc antibody and wt Gag or chimeric proteins were detected with an anti-p24CA antibody

Fig. 5

Fig. 5

Incorporation of GST fusion proteins into virus-like particles. 293T cells were co-transfected with 10 μg of NC(N1) (panel a) or NC(N2) (panel b) plus 10 μg pBlueScript SK or indicated construct. At 48–72 h post-transfection, supernatant and cells were collected, prepared, and subjected to SDS-10% PAGE. HIV-1 Gag or chimeric proteins were detected by Western immunoblotting using a monoclonal antibody against HIV-1 p24CA. GST or GST fusions were probed with an anti-GST monoclonal antibody

Fig. 6

Fig. 6

Membrane flotation analysis of chimeric proteins. 293T cells were transfected with wt or indicated expression construct. (a) Membrane binding-defective Gag (nonmyristylated, Myr−) served as control. Cells were harvested, homogenized, and subjected to equilibrium flotation centrifugation analysis 2 days post-transfection. Ten fractions were collected from top to bottom. Fraction aliquots were resolved using SDS-PAGE (10%) and probed with a monoclonal antibody directed against HIV-1 CA or the Myc tag. During ultracentrifugation, membrane-bound Gag proteins floated to the 10–65% sucrose interface and became enriched in fraction 2. (b) Band densities in fractions 2, 3 and 4 were divided by total band densities of fractions 1–10 and multiplied by 100 to obtain percentages of membrane-bound protein for each construct

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