Basic residues of the retroviral nucleocapsid play different roles in gag-gag and Gag-Psi RNA interactions.
The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal (Psi)-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored Psi RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for Psi RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.
Lee
Eun-Gyung
EG
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109-1024, USA.
Linial
Maxine L
ML
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019251:Nucleocapsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D015683:Gene Products, gag; D012367:RNA, Viral