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Journal of Virology, January 2009, p. 802-810, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01521-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA {triangledown} ,{dagger}

Tayyba T. Baig, Jean-Marc Lanchy, and J. Stephen Lodmell*

Division of Biological Sciences, The University of Montana, Missoula, Montana 59812

Received 18 July 2008/ Accepted 23 October 2008

The packaging signal ({psi}) of human immunodeficiency virus type 2 (HIV-2) is present in the 5' noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5'-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3' side of pal (GCUCC-3') is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5' side of pal (5'-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5' side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5' side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.

* Corresponding author. Mailing address: Division of Biological Sciences, The University of Montana, Science Complex, Rm. 202, Missoula, MT 59812. Phone: (406) 243-6393. Fax: (406) 243-4304. E-mail: stephen.lodmell{at}umontana.edu

{triangledown} Published ahead of print on 29 October 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, January 2009, p. 802-810, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01521-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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