Biarsenical labeling of tetracysteine-tagged proteins for tracking existing and newly synthesized pools of proteins (original) (raw)
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Journal of Virology, 2010
Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs, resulting in the formation of multiprotein complex. Our results further show that the GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of the CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell.
Journal of General Virology, 2001
Glycoprotein 5 (GP5) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). Expression of GP5 has been improved by removing the transmembrane regions. Vectors were constructed encoding complete GP5 plus three mutants: GP5 ΔNs (residues 28–201), GP5[30–67] (residues 30–67) and GP5[30–201] (residues 30–67/130–201). The three deletion mutants were expressed at levels 20–30 times higher than complete GP5. GP5[30–201] was well recognized in ELISA or immunoblotting by a collection of pig sera. All the fragments were tested for the generation of MAbs, but only the polyhistidine-tagged fragment GP5[30–201]H elicited an antibody response sufficient to produce MAbs. The two MAbs were positive for PRRSV in ELISA and immunoblotting, but negative for virus neutralization. MAb 4BE12 reacted with residues 130–170 and MAb 3AH9 recognized residues 170–201. This region was recognized strongly in immunoblotting by a collection of infected-pig sera. These results indi...
Virology, 2012
The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω − 2, where ω is the GPI moiety at E160), P159 (ω − 1), and M162 (ω + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to colocalize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.
Journal of Virology, 2005
Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP 5 and M and the minor proteins GP 2a , E, GP 3 , and GP 4 . Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP 5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indi...
Journal of Virology, 1998
The GP 3 protein of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in 293 cells by a recombinant human type 5 adenovirus carrying the open reading frame 3 gene. The protein exhibited a molecular mass of 42 kDa and comigrated with GP 3 expressed in PRRSV-infected MARC-145 cells. Removal of N-linked glycans from GP 3 resulted in a 27-kDa protein (P3), confirming its highly glycosylated nature. Pulse-chase experiments carried out either in the context of PRRSV infection or upon individual expression of GP 3 in 293 cells showed that the protein remains completely endo-β- N -acetylglucosaminidase H-sensitive even after 4 h of synthesis. Thus, the transport of GP 3 was restricted to the premedial Golgi compartment, presumably the endoplasmic reticulum (ER). However, a minor fraction of GP 3 was found to be secreted in the culture medium as a soluble membrane-free form. This released protein (sGP 3 ) was readily identified upon individual e...
Journal of Virology, 2006
Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.
Virology, 1996
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and E. The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated. Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively. In the present report, monospecific antisera raised against Escherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line. Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans. Pulse-chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartment. During the first 30 min of chase, E undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures. Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated. These two differentially glycosylated forms of E were found to be incorporated into extracellular virions. In cells and virions, both M and E were shown to occur in heterodimeric complexes linked by disulfide bonds. The oligomerization process, as analyzed from pulse-chase experiments, showed that M and E are incorporated into ME complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus. Apparently, all steps of E protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER). In the infected cells, E and M appear highly membrane-associated, while N is predominantly cytosolic.
Archives of Virology, 1998
Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reversetranscribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M r) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP 3) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP 3 protein was present in amounts equivalent to those of the N, M, and GP 5 proteins in the infected cells, whereas no GP 3 could be detected in purified virions. During the first 30 min of chase, the GP 3 undergoes a gradual downward shift of its apparent M r , thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural.
Archives of Virology, 1999
The identification of antigens recognized by T cell responses has become fundamental for developing effective immunizations against viral infections. Lymphocyte proliferation and delayed-type hypersensitivity responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection have been demonstrated. However, the polypeptide specificity of T cell responses to PRRSV is unknown. To identify the PRRSV polypeptides recognized by porcine lymphocytes two approaches were employed. First polypeptides of purified virions were separated by SDS-PAGE and particle suspensions obtained from nitrocellulose blots were used as antigens. Second, the polypeptides encoded by ORFs 2, 4, 5, 6, and 7 of the strain VR-2 332 were expressed as fusion proteins with a histidine tag in mammalian cells, using vaccinia virus as expression system. Significant antigen-specific proliferation responses to the matrix and envelope proteins from purified virions were obtained. This finding was supported by specific and dose-dependent proliferation responses to the recombinant polypeptides encoded by ORF2, 5 and 6 detected in virus-infected but not in control pigs. These results demonstrate that T-cell responses can be detected to individual PRRSV polypeptides. The greater response to the product of ORF6 than to the other PRRSV polypeptides indicates that the viral matrix polypeptide may have a major role in cellular immunity.