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Journal of Virology, November 2003, p. 11973-11984, Vol. 77, No. 22
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.22.11973-11984.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Primary Sequence and Secondary Structure Motifs in Spleen Necrosis Virus RU5 Confer Translational Utilization of Unspliced Human Immunodeficiency Virus Type 1 Reporter RNA

Tiffiney M. Roberts1,2 and Kathleen Boris-Lawrie1,2,3,4,5*

Center for Retrovirus Research,1 Departments of Veterinary Biosciences,4 Molecular Virology, Immunology & Medical Genetics,2 Molecular, Cellular & Developmental Biology Graduate Program,3 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210-10935

Received 23 June 2003/ Accepted 20 August 2003

The 5' long terminal repeat (LTR) of spleen necrosis virus (SNV) contains a unique posttranscriptional control element that facilitates Rev/Rev-responsive element-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA. HIV-1 Gag expression is eliminated when SNV LTR is repositioned to the 3' untranslated region or when the RU5 region is positioned in the antisense orientation. RU5 corresponds to the 5' RNA terminus, and results presented here indicate that Gag production is sustained upon introduction of transcribed spacers that reposition SNV RU5 35 to 200 nucleotides downstream. Concordant results of deletion and point mutagenesis identified two functionally redundant and synergistic motifs (designated A and C) that are necessary and sufficient for SNV RU5 activity. Enzymatic analysis of SNV RU5 RNA structure determined that A and C correspond to stem-loop structures. Quantitative RNA and protein analysis of A and C mutants revealed that the structural integrity of A and C is necessary for protein production, and loss of function correlates with little change in steady-state level, splicing efficiency, or cytoplasmic accumulation of HIV-1 gag reporter RNA. Instead, the structural mutations eliminate cytoplasmic utilization as an mRNA template for Gag protein production. Point mutations of unpaired loop-and-bulge nucleotides that maintain the structure of A eliminate activity. The results show that the unpaired UUGU loop and U-rich bulges function together and are candidate SNV RU5 binding sites for the host cell protein(s) that directs cytoplasmic utilization of unspliced HIV-1 reporter RNA.

* Corresponding author. Mailing address: Goss Laboratory, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Phone: (614) 292-1392. Fax: (614) 292-6473. E-mail: boris-lawrie.1{at}osu.edu.

Journal of Virology, November 2003, p. 11973-11984, Vol. 77, No. 22
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.22.11973-11984.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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