Poster abstracts

Poster number 5 submitted by Erik Olson

Structure and specificity of Gag-gRNA complexes provide insights into mechanism of selective genome packaging in HIV-1

Erik D Olson (Ohio State Biochemistry Program, Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), William A Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Tiffiny Rye-McCurdy (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Brian R Thompson (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Ioulia Rouzina (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH)

Abstract:
In infected cells, host RNA is present in vast excess to viral RNA, yet all retroviruses specifically package their full-length, dimeric, genomic RNA (gRNA). The psi element within the gRNA 5ยด-untranslated region are critical for specific Gag-gRNA interactions. However, under physiological conditions, in vitro binding assays are not consistent with a significantly higher Gag binding affinity for psi over non-psi RNA. Thus, based on binding affinity alone, Gag is not likely to achieve levels of discrimination sufficient to account for the high selectivity of gRNA packaging. To determine whether this selectivity could be detected using a different approach, we developed a salt-titration assay, which allows us to determine two binding parameters: Kd(1M), which describes the non-electrostatic strength of binding, and Zeff, the effective positive charge of the protein-RNA interaction.
Compared to other RNAs tested, HIV-1 Gag binds to psi RNA with lower Kd(1M) and Zeff, suggesting that Gag-psi binding selectivity is based on specific interactions. A Gag mutant lacking MA bound both psi and other RNAs with similar Zeff and Kd(1M) values, indicating that the MA domain mediates the increased electrostatic interactions observed in Gag-non-psi RNA binding, and contributes to the ability of Gag to select psi over non-psi RNA. We are currently dissecting the elements of psi responsible for this specific binding interaction.
We have also studied Rous sarcoma virus (RSV) Gag-RNA interactions. Similar to HIV-1, we found that RSV Gag-psi binding was less electrostatic and more specific than binding to non-psi RNA. These differences were largely eliminated in the absence of MA. Small-angle X-ray scattering analysis of HIV-1 and RSV psi RNAs revealed a remarkably similar global fold, suggesting that even in the absence of sequence similarity, the mechanism of Gag-psi binding may be conserved among retroviruses.

Keywords: HIV , RNA