Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies

Gretchen B Caughman, J. B. Lewis, R. H. Smith, R. N. Harty, D. J. O'Callaghan

Research output: Contribution to journalArticle

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Abstract

During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro- translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.

Original languageEnglish (US)
Pages (from-to)3024-3032
Number of pages9
JournalJournal of Virology
Volume69
Issue number5
StatePublished - Jan 1 1995

Fingerprint

Equid Herpesvirus 1
Equid herpesvirus 1
monoclonal antibodies
Monoclonal Antibodies
Genes
Immediate-Early Proteins
Proteins
genes
proteins
Viral DNA
Infection
Virion
virion
Epitopes
infection
epitopes
Amino Acids
promoter regions
Nucleocapsid
Messenger RNA

ASJC Scopus subject areas

  • Immunology

Cite this

Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies. / Caughman, Gretchen B; Lewis, J. B.; Smith, R. H.; Harty, R. N.; O'Callaghan, D. J.

In: Journal of Virology, Vol. 69, No. 5, 01.01.1995, p. 3024-3032.

Research output: Contribution to journalArticle

Caughman, Gretchen B ; Lewis, J. B. ; Smith, R. H. ; Harty, R. N. ; O'Callaghan, D. J. / Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies. In: Journal of Virology. 1995 ; Vol. 69, No. 5. pp. 3024-3032.
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abstract = "During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro- translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.",
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AU - Caughman, Gretchen B

AU - Lewis, J. B.

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AU - Harty, R. N.

AU - O'Callaghan, D. J.

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AB - During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro- translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.

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