The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame

Prashant Desai, Simon C. Watkins, Stanley Person

Research output: Contribution to journalArticle

Abstract

Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N- terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26ΔZ, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26ΔZ that sedimented more rapidly than B capsids from wild-type- infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells. Therefore, 22a and perhaps the less abundant 21 form a scaffold that is essential for B capsid formation and its icosahedral symmetry. Since several bacteriophages employ a scaffold to determine capsid size and shape, it appears that HSV-1 uses the same principles for capsid assembly and maturation. The size and symmetry of HSV-1 capsids must require the interaction of 22a and perhaps 21 with one or more of the remaining four components of the capsids. In related experiments a thiol-cleavable cross-linking reagent, dithiobis (succinimidylpropionate), was used to test for cross-links in the molecules that make up B capsids. Protein 22a formed cross-links to itself and to VP5 and VP19C, which may be indicative of the interactions required for the icosahedral symmetry of HSV-1 capsids. Cross-links were also detected between VP5, VP19C, and VP23. Null mutant viruses for VP5 or VP23 do not form recognizable structures in nonpermissive cells, and the mutants retain the protease-processing activity associated with the UL26 gene product. Although the enzyme activity of the UL26 gene product may be independent of capsid structure, the location of VP24 and 21 within wild-type capsids suggests a role for the protease in capsid maturation.

Original languageEnglish (US)
Pages (from-to)5365-5374
Number of pages10
JournalJournal of Virology
Volume68
Issue number9
StatePublished - Sep 1994
Externally publishedYes

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Human herpesvirus 1
capsid
Capsid
Human Herpesvirus 1
Open Reading Frames
open reading frames
Genes
genes
Peptide Hydrolases
proteinases
mutants
Vero Cells
Viruses
viruses
cells
Mutation
Growth
Proteins
virion
proteins

ASJC Scopus subject areas

  • Immunology

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The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame. / Desai, Prashant; Watkins, Simon C.; Person, Stanley.

In: Journal of Virology, Vol. 68, No. 9, 09.1994, p. 5365-5374.

Research output: Contribution to journalArticle

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title = "The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame",
abstract = "Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N- terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26ΔZ, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26ΔZ that sedimented more rapidly than B capsids from wild-type- infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells. Therefore, 22a and perhaps the less abundant 21 form a scaffold that is essential for B capsid formation and its icosahedral symmetry. Since several bacteriophages employ a scaffold to determine capsid size and shape, it appears that HSV-1 uses the same principles for capsid assembly and maturation. The size and symmetry of HSV-1 capsids must require the interaction of 22a and perhaps 21 with one or more of the remaining four components of the capsids. In related experiments a thiol-cleavable cross-linking reagent, dithiobis (succinimidylpropionate), was used to test for cross-links in the molecules that make up B capsids. Protein 22a formed cross-links to itself and to VP5 and VP19C, which may be indicative of the interactions required for the icosahedral symmetry of HSV-1 capsids. Cross-links were also detected between VP5, VP19C, and VP23. Null mutant viruses for VP5 or VP23 do not form recognizable structures in nonpermissive cells, and the mutants retain the protease-processing activity associated with the UL26 gene product. Although the enzyme activity of the UL26 gene product may be independent of capsid structure, the location of VP24 and 21 within wild-type capsids suggests a role for the protease in capsid maturation.",
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T1 - The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame

AU - Desai, Prashant

AU - Watkins, Simon C.

AU - Person, Stanley

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N2 - Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N- terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26ΔZ, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26ΔZ that sedimented more rapidly than B capsids from wild-type- infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells. Therefore, 22a and perhaps the less abundant 21 form a scaffold that is essential for B capsid formation and its icosahedral symmetry. Since several bacteriophages employ a scaffold to determine capsid size and shape, it appears that HSV-1 uses the same principles for capsid assembly and maturation. The size and symmetry of HSV-1 capsids must require the interaction of 22a and perhaps 21 with one or more of the remaining four components of the capsids. In related experiments a thiol-cleavable cross-linking reagent, dithiobis (succinimidylpropionate), was used to test for cross-links in the molecules that make up B capsids. Protein 22a formed cross-links to itself and to VP5 and VP19C, which may be indicative of the interactions required for the icosahedral symmetry of HSV-1 capsids. Cross-links were also detected between VP5, VP19C, and VP23. Null mutant viruses for VP5 or VP23 do not form recognizable structures in nonpermissive cells, and the mutants retain the protease-processing activity associated with the UL26 gene product. Although the enzyme activity of the UL26 gene product may be independent of capsid structure, the location of VP24 and 21 within wild-type capsids suggests a role for the protease in capsid maturation.

AB - Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N- terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26ΔZ, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26ΔZ that sedimented more rapidly than B capsids from wild-type- infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells. Therefore, 22a and perhaps the less abundant 21 form a scaffold that is essential for B capsid formation and its icosahedral symmetry. Since several bacteriophages employ a scaffold to determine capsid size and shape, it appears that HSV-1 uses the same principles for capsid assembly and maturation. The size and symmetry of HSV-1 capsids must require the interaction of 22a and perhaps 21 with one or more of the remaining four components of the capsids. In related experiments a thiol-cleavable cross-linking reagent, dithiobis (succinimidylpropionate), was used to test for cross-links in the molecules that make up B capsids. Protein 22a formed cross-links to itself and to VP5 and VP19C, which may be indicative of the interactions required for the icosahedral symmetry of HSV-1 capsids. Cross-links were also detected between VP5, VP19C, and VP23. Null mutant viruses for VP5 or VP23 do not form recognizable structures in nonpermissive cells, and the mutants retain the protease-processing activity associated with the UL26 gene product. Although the enzyme activity of the UL26 gene product may be independent of capsid structure, the location of VP24 and 21 within wild-type capsids suggests a role for the protease in capsid maturation.

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