title: Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging.
abstract: The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle.
  
  
  OBJECTIVE
  Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Prague, Czech Republic.
authors.forename: Tibor
authors.initials: T
authors.lastname: Füzik
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Prague, Czech Republic.
authors.forename: Růžena
authors.initials: R
authors.lastname: Píchalová
authors.affiliation: Structural and Computational Biology Unit and Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Florian K M
authors.initials: FKM
authors.lastname: Schur
authors.affiliation: Institute of Organic Chemistry and Biochemistry IOCB Research Centre and Gilead Sciences, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Karolína
authors.initials: K
authors.lastname: Strohalmová
authors.affiliation: Institute of Organic Chemistry and Biochemistry IOCB Research Centre and Gilead Sciences, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Ivana
authors.initials: I
authors.lastname: Křížová
authors.affiliation: Institute of Organic Chemistry and Biochemistry IOCB Research Centre and Gilead Sciences, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Romana
authors.initials: R
authors.lastname: Hadravová
authors.affiliation: Institute of Organic Chemistry and Biochemistry IOCB Research Centre and Gilead Sciences, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Rumlová
authors.affiliation: Structural and Computational Biology Unit and Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: John A G
authors.initials: JAG
authors.lastname: Briggs
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Prague, Czech Republic ulbrichp@vscht.cz rumlt@vscht.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Ulbrich
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Prague, Czech Republic ulbrichp@vscht.cz rumlt@vscht.cz.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Ruml
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D020285:Cryoelectron Microscopy; D015683:Gene Products, gag; D016679:Genome, Viral; D006801:Humans; D016093:Mason-Pfizer monkey virus / Q000502:physiology* / Q000648:ultrastructure; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins; D019065:Virus Assembly / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D015683:Gene Products, gag; D012367:RNA, Viral; D011994:Recombinant Proteins