Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus.
The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N(TAIL)). XD binding induces N(TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced N(TAIL) folding, XD-N(TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N(TAIL) α-helical folding were created within Box-2 of Edmonston MeV N(TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N(TAIL) binding affinity or reduction/loss of XD-induced N(TAIL) alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.
Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio 43210, USA.
Yaoling
Y
Shu
Johnny
J
Habchi
Stéphanie
S
Costanzo
André
A
Padilla
Joanna
J
Brunel
Denis
D
Gerlier
Michael
M
Oglesbee
Sonia
S
Longhi
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008459:Measles virus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic; D014709:Vero Cells; D019562:Viral Load; D014771:Virion / Q000378:metabolism; D014779:Virus Replication
D046912:Multiprotein Complexes; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases