title: Conserved determinants of lentiviral genome dimerization.
abstract: BACKGROUND
  Retroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by "kissing" interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5' element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5' leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy.
  
  
  RESULTS
  Dimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg(2+), indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers.
  
  
  CONCLUSIONS
  The finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5' leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. lphthaotran@umbc.edu.
authors.forename: Thao
authors.initials: T
authors.lastname: Tran
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. liu5@umbc.edu.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. janm@umbc.edu.
authors.forename: Jan
authors.initials: J
authors.lastname: Marchant
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. samon1@umbc.edu.
authors.forename: Sarah
authors.initials: S
authors.lastname: Monti
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. smichel1@umbc.edu.
authors.forename: Michelle
authors.initials: M
authors.lastname: Seu
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. jzaki1@umbc.edu.
authors.forename: Jessica
authors.initials: J
authors.lastname: Zaki
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. op19496@umbc.edu.
authors.forename: Ae Lim
authors.initials: AL
authors.lastname: Yang
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. jenbohn@umich.edu.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bohn
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. venkat2@umbc.edu.
authors.forename: Venkateswaran
authors.initials: V
authors.lastname: Ramakrishnan
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. rashmi1@umbc.edu.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Singh
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. mateoh@mail.usf.edu.
authors.forename: Mateo
authors.initials: M
authors.lastname: Hernandez
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. av14@students.uwf.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Vega
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA. summers@hhmi.umbc.edu.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Summers
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D019281:Dimerization; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D015498:HIV-2 / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D009690:Nucleic Acid Conformation; D019251:Nucleocapsid / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D015302:Simian Immunodeficiency Virus / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral