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The measles virus matrix gene and gene product defined by in vitro and in vivo expression
Abstract:
Sequence analysis of full-length cDNA clones of the measles virus matrix gene revealed three possible open reading frames: M, X1, and X2. The M reading frame differed from the reported sequence by a single nucleotide corresponding to a conservative lysine to arginine amino acid substitution near the carboxy-terminus conserved among the M proteins of paramyxoviruses. The putative X reading frames contained no translational termination codon due to a frame-shift mutation. The protein-coding potential of these reading frames was examined by in vitro translation and DNA-mediated gene transfer into primate cells. The M reading frame produced a 38,000 Mr protein indistinguishable from the M protein in measles virus-infected cells. This protein was not phosphorylated nor processed post-translationally in vivo. The putative X1 and X2 reading frames could be translated into proteins when placed near the 5' terminus of the RNA. The resulting proteins were heterogeneous due to the lack of a termination codon. Translation from the putative X reading frames was adversely affected by an upstream AUG codon and these reading frames were unable to synthesize proteins in their normal 3' locations. At least 146 nucleotides of these 3'-untranslated sequences could be deleted without affecting the expression of M protein in vitro or in vivo. Thus despite the multiple open reading frames, the measles virus M gene is functionally monocistronic.
Insights
The measles virus matrix (M) gene primarily produces one functional protein, M, despite containing multiple potential reading frames. Other frames (X1, X2) show limited protein synthesis due to mutations and location.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- The measles virus matrix (M) gene is crucial for viral assembly and budding.
- Previous studies suggested potential for multiple protein products from the M gene.
- Understanding the M gene's coding potential is key to measles virus replication.
Purpose of the Study:
- To investigate the protein-coding potential of all identified open reading frames (ORFs) within the measles virus matrix gene.
- To determine the functional significance of the M, X1, and X2 reading frames.
- To clarify the monocistronic or polycistronic nature of the measles virus M gene.
Main Methods:
- Sequence analysis of full-length measles virus M gene cDNA clones.
- In vitro translation assays to assess protein synthesis from different ORFs.
- DNA-mediated gene transfer into primate cells to study protein expression in vivo.
- Site-directed mutagenesis to investigate the effect of upstream AUG codons and 3'-untranslated sequences.
Main Results:
- Three potential open reading frames (M, X1, X2) were identified in the measles virus M gene.
- The M reading frame produced a 38,000 Mr protein identical to the native M protein, which was not phosphorylated or post-translationally modified.
- The X1 and X2 reading frames showed limited and heterogeneous protein synthesis, affected by an upstream AUG codon and their 3' location.
- Deletion of up to 146 nucleotides from the 3'-untranslated region did not impact M protein expression.
Conclusions:
- The measles virus M gene functions as a monocistronic unit, primarily expressing the M protein.
- The X1 and X2 reading frames have minimal or no functional role in protein synthesis under normal conditions.
- The identified sequence variations in the M reading frame represent conservative amino acid substitutions.