RNA-binding proteins of coronavirus MHV: Detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay

Robbins, Susan G.; Frana, Mark F.; McGowan, John J.; Boyle, John; Holmest, Kathryn V.

doi:10.1016/0042-6822(86)90305-3

Cited by 81 publications

(88 citation statements)

References 41 publications

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“…5). The smaller bands which migrated slightly ahead of the N protein may correspond to degradation products, as previously described with some other coronaviruses (Siddell et al, 1981 ;Robbins et al, 1986;. With purified viral preparations, a non-specific background of N protein was occasionally observed when anti-E2 and anti-E3 MAbs were tested, both by Western immunoblotting and immunoprecipitation experiments.…”

Section: Discussionmentioning

confidence: 78%

Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

Dea

Tijssen

1989

Journal of General Virology

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SUMMARYTwenty-nine hybridoma cell lines, producing monoclonal antibodies (MAbs) to the Minnesota strain of turkey enteric coronavirus (TCV), have been established by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the egg-adapted or tissue culture-adapted virus. The hybridomas produced mainly IgG2a or IgG1 antibodies. Western immunoblotting experiments with purified virus, and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts, allowed assessment of the polypeptide specificity of the MAbs. Sixteen hybridomas secreted antibodies directed to the peplomeric protein (E2, gp200/gpl00) and putative intracellular precursors of apparent Mr 170K to 180K and 90K. Four hybridomas produced antibodies that selectively reacted with a glycoprotein with an Mr of 140K (E3). This polypeptide species corresponded to the major structural component of small granular projections, located near the base of the larger bulbous peplomers, and was found to be responsible for haemagglutination. The major neutralization-mediating determinants were found to be carried by both E2 and E3 glycoproteins. Eight hybridomas produced MAbs directed to the major nucleocapsid protein (N, 52K), and only one MAb reacted with a low Mr structural glycoprotein (24K), corresponding to the matrix (El) protein. By indirect immunofluorescence, MAbs of different specificity also revealed distinct patterns of distribution of the viral antigens within the cells. The location on the virion of the antigenic determinants recognized by MAbs of different specificity was determined by the use of an immunogold electron microscopy technique. Comparison of nine TCV Quebec strains, using MAbs directed to peplomer and haemagglutinin proteins of the prototype Minnesota strain, confirmed their close antigenic relationship, but also revealed the occurrence of at least two distinct antigenic groups.

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Section: Discussionmentioning

confidence: 78%

Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

Dea

Tijssen

1989

Journal of General Virology

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“…Accordingly, the NЈ form of the MHV N protein was shown to conserve its RNA binding properties (30). The function of the acidic carboxy-terminal domain of coronavirus N protein remains unknown (19).…”

Section: Discussionmentioning

confidence: 99%

The Viral Nucleocapsid Protein of Transmissible Gastroenteritis Coronavirus (TGEV) Is Cleaved by Caspase-6 and -7 during TGEV-Induced Apoptosis

Éléouët

Slee

Saurini

et al. 2000

J Virol

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The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and ␣-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD 359 2. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.

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“…Using a filter-binding assay, Huismans and Joklik (27) (9,29,36) and viral and cellular RNA binding proteins (10,47,49). The optimal conditions determined for cr3 dsRNA binding in this assay (pH 6.4 to 6.8; 0 to 50 mM NaCI) were very similar to those for the RNA binding of rotavirus VP2 (10) and the DNA binding of bovine papillomavirus E2 protein (36), both of which have been characterized as sequence-independent interactions.…”

Section: Discussionmentioning

confidence: 99%

Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

Schiff¹,

Nibert²,

Co³

et al. 1988

Mol. Cell. Biol.

122

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By atomic absorption analysis, we determined that the reovirus outer capsid protein cr3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc-and dsRNA-binding activities of cr3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxyterminal fragment. By these techniques, new zinc-and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor lIlA-Like zinc-binding site within a3. We suggest that the zinc-and dsRNA-binding activities of cr3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.The mammalian reoviruses represent the prototype of a group of nonenveloped plant and animal viruses whose segmented, double-stranded RNA (dsRNA) genomes are surrounded by an inner capsid core and an outer capsid shell. Three serotypes of mammalian reoviruses have been identified, and the different strains of these viruses studied to date have been found to vary with respect to a number of biochemical markers and biological properties. As a result of these polymorphisms, reoviruses offer an excellent model system for the study of virion morphogenesis and virus-host cell interactions because the genetic analysis of reassortant viruses has provided a means to assign biological phenotypes and biochemical properties to particular viral genome segments. Results of studies with reassortant viruses have suggested that, in addition to playing structural roles, the reovirus outer capsid proteins influence the virus-host cell interaction at a variety of stages in the viral replicative cycle. S4, the smallest of the 10 mammalian reovirus genes, encodes the a3 protein (38, 40), a major component of the virion outer capsid (53). The or3 protein is the only reovirus protein that has been reported to have affinity for dsRNA (27). This is an unusual property for an outer capsid protein which is removed from infecting parental virus by protease digestion early in infection (52, 55), and suggests that this property may be one of newly synthesized a3 that operates in the cell cytoplasm. Results of biochemical and genetic studies have implied a role for the S4 gene product in the regulation of viral transcription (4) and translation (33, 34), the inhibition of host cell RNA and protein synthesis (50), and the establishment of persistent reovirus infection (1). To begin to determine whether the ability of cr3 to bind to dsRNA is mechanistically important for virion morphogenesis or any of the biological properties which have been mapped to the S4 gene segment, we initiated a study to investigate the structural basis for the dsRNA-binding activity of cr3. * Corresponding author. A re...

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RNA-binding proteins of coronavirus MHV: Detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay

Cited by 81 publications

References 41 publications

Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

The Viral Nucleocapsid Protein of Transmissible Gastroenteritis Coronavirus (TGEV) Is Cleaved by Caspase-6 and -7 during TGEV-Induced Apoptosis

Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

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