Amino acids encoded downstream of gag are not required by rous sarcoma virus protease during gag-mediated assembly

R. P. Bennett, S. Rhee, R. C. Craven, E. Hunter, J. W. Wills

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the e protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.

Original languageEnglish (US)
Pages (from-to)272-280
Number of pages9
JournalJournal of virology
Volume65
Issue number1
StatePublished - Jan 1 1991

Fingerprint

Rous sarcoma virus
proteinases
Amino Acids
Reading Frames
amino acids
Peptide Hydrolases
ribosomes
Ribosomes
Retroviridae
nonsense mutation
mutation
mutants
Mutation
Rous sarcoma virus protease
RNA-directed DNA polymerase
Nonsense Codon
RNA-Directed DNA Polymerase
Capsid Proteins
coat proteins
virion

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

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title = "Amino acids encoded downstream of gag are not required by rous sarcoma virus protease during gag-mediated assembly",
abstract = "Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the e protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.",
author = "Bennett, {R. P.} and S. Rhee and Craven, {R. C.} and E. Hunter and Wills, {J. W.}",
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Amino acids encoded downstream of gag are not required by rous sarcoma virus protease during gag-mediated assembly. / Bennett, R. P.; Rhee, S.; Craven, R. C.; Hunter, E.; Wills, J. W.

In: Journal of virology, Vol. 65, No. 1, 01.01.1991, p. 272-280.

Research output: Contribution to journalArticle

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T1 - Amino acids encoded downstream of gag are not required by rous sarcoma virus protease during gag-mediated assembly

AU - Bennett, R. P.

AU - Rhee, S.

AU - Craven, R. C.

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AU - Wills, J. W.

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N2 - Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the e protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.

AB - Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the e protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.

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