trans-acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants

R. A. Garbitt, J. A. Albert, M. D. Kessler, L. J. Parent

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E-). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E-.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E-, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG- was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG-, Myr1E-, and Myr1E-.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.

Original languageEnglish (US)
Pages (from-to)260-268
Number of pages9
JournalJournal of virology
Volume75
Issue number1
DOIs
StatePublished - Jan 1 2001

Fingerprint

Rous sarcoma virus
dimerization
Dimerization
RNA
genomics
mutants
Mutant Proteins
Proteins
proteins
Viral Matrix Proteins
gag Gene Products
Viral RNA
nucleotide sequences
Retroviridae
Amino Acid Substitution
Green Fluorescent Proteins
Virion
pathogenicity
Nucleotides
Maintenance

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Garbitt, R. A. ; Albert, J. A. ; Kessler, M. D. ; Parent, L. J. / trans-acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants. In: Journal of virology. 2001 ; Vol. 75, No. 1. pp. 260-268.
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trans-acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants. / Garbitt, R. A.; Albert, J. A.; Kessler, M. D.; Parent, L. J.

In: Journal of virology, Vol. 75, No. 1, 01.01.2001, p. 260-268.

Research output: Contribution to journalArticle

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T1 - trans-acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants

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N2 - The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E-). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E-.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E-, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG- was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG-, Myr1E-, and Myr1E-.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.

AB - The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E-). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E-.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E-, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG- was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG-, Myr1E-, and Myr1E-.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.

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