High Affinity Nucleocapsid Protein Binding to the μΨ RNA Packaging Signal of Rous Sarcoma Virus

• Published in: Journal of molecular biology Vol. 349; no. 5; pp. 976 - 988
• Main Authors: Zhou, Jing, McAllen, John K., Tailor, Yogita, Summers, Michael F.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 24.06.2005
• Subjects: nuclear magnetic resonance (NMR); nucleocapsid protein; psi-site; ribonucleic acid (RNA); Rous sarcoma virus; HIV-1; PBS; A; ORF; psi-site; ACE; C; ribonucleic acid (RNA); G; Rous sarcoma virus; GST; SD; UTR; RSV; NC; U; nuclear magnetic resonance (NMR); MLV; nucleocapsid protein; ITC; BME; PCR
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2005.04.046

The genomes of all retroviruses contain sequences near their 5′ ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called μΨ, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the μΨ encapsidation element. NC does not bind with significant affinity to truncated forms of μΨ, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native μΨ RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC–RNA complexes (extrapolated dissociation constant K d=1.9 nM). Tight binding with 1:1 NC-μΨ stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC–RNA binding differ considerably from those observed for other retroviruses.