title: Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication.
abstract: The 5' UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 5' untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The "winner" viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication.
authors: Division of Biological Sciences, The University of Montana, Missoula, Montana 59812, USA.
authors: Christy L
authors: CL
authors: Strong
authors: Jean-Marc
authors: JM
authors: Lanchy
authors: J Stephen
authors: JS
authors: Lodmell
references: Nucleic Acids Res. 2004 Aug 27;32(15):4585-95
references: 15333691
references: Biochemistry. 2008 Mar 11;47(10):3283-93
references: 18278873
references: J Mol Biol. 2009 Aug 14;391(2):426-37
references: 19559029
references: Nat Rev Microbiol. 2005 Aug;3(8):643-55
references: 16064056
references: Biochemistry. 2003 Mar 11;42(9):2634-42
references: 12614158
references: Nucleic Acids Res. 1993 Nov 11;21(22):5020-4
references: 8255755
references: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references: 12824337
references: Nucleic Acids Res. 2011 Jul;39(12):5232-44
references: 21393254
references: J Virol. 1990 Sep;64(9):4207-16
references: 2384918
references: J Biol Chem. 2003 Mar 28;278(13):11601-11
references: 12458192
references: Biochemistry. 2008 Dec 9;47(49):13064-73
references: 19006324
references: Proc Natl Acad Sci U S A. 1989 Aug;86(15):5781-5
references: 2788277
references: Trends Biochem Sci. 1998 Aug;23(8):297-301
references: 9757830
references: J Virol. 1992 Aug;66(8):4824-33
references: 1629957
references: J Virol. 2007 May;81(9):4664-76
references: 17329338
references: Nature. 2004 Sep 30;431(7008):586-90
references: 15457265
references: Nucleic Acids Res. 1987 Oct 26;15(20):8125-48
references: 3313277
references: J Virol. 2009 Jan;83(2):802-10
references: 18971263
references: Nucleic Acids Res. 2002 Jun 15;30(12):2647-55
references: 12060681
references: Biochemistry. 2005 Jun 28;44(25):9058-66
references: 15966729
references: Nat Struct Mol Biol. 2005 Nov;12(11):1001-7
references: 16244661
references: AIDS Res Hum Retroviruses. 1993 Apr;9(4):331-5
references: 8512748
references: Cell. 1986 Jan 31;44(2):283-92
references: 3943125
references: RNA. 2008 Jul;14(7):1443-55
references: 18495939
references: PLoS Biol. 2008 Apr 29;6(4):e96
references: 18447581
references: Retrovirology. 2008 Jul 17;5:65
references: 18637186
references: Prog Nucleic Acid Res Mol Biol. 1996;54:1-34
references: 8768071
references: Mol Cell Biol. 1992 Jan;12(1):207-19
references: 1729599
references: Virology. 2007 Jul 5;363(2):261-71
references: 17331561
references: Nucleic Acids Res. 2010 Mar;38(4):1367-81
references: 19969542
references: RNA. 2003 Aug;9(8):1007-18
references: 12869711
references: J Mol Biol. 2004 Feb 13;336(2):369-79
references: 14757051