HIV-1 is a retrovirus that uses RNA as its genomic material. The RNA must be reverse transcribed to DNA prior to integration into human DNA. New viral RNAs are transcribed, exported from the nucleus, and selectively packaged into new viral particles. I am currently working on two projects to improve our understanding of how HIV-1 is able to selectively recognize and package its genomic RNA over the vast excess of other cellular RNAs.
The first ~350 nucleotides of the HIV-1 viral RNA genome (vRNA), a region known as the 5´ untranslated region (5´UTR), is responsible for regulating numerous aspects of the viral life cycle. This functionally dense RNA is capable of this regulation due in large part to its higher order structure, and has been the subject of many secondary structural probing studies . There have also been several 3D structures of individual hairpins determined by NMR, but a tertiary structure of the entire 5´UTR remains to be elucidated . To achieve this goal I am working on characterizing the tertiary structure of the entire 5´UTR using small angle X-ray scattering (SAXS) .
A region of the 5´UTR, the so-called Psi packaging signal, has been shown to play an essential role in directing the correct vRNA to be packaged into budding virions , although further downstream elements have been implicated in packaging as well . Correct recognition of the vRNA is mediated via interactions with the NC domain of the viral Gag protein . The study of Gag-RNA interactions is complicated by Gag’s ability to interact with nucleic acids nonspecifically, in addition to its specific interaction with Psi. We have previously applied a salt-titration assay that distinguishes between Gag binding in these two distinct modes, and have determined the contribution of various Gag domains to vRNA recognition . My goal is to use this assay to test numerous RNA variants of Psi in the context of smaller RNAs, as well as in the entire 5´UTR, to identify the sequence and structural elements that confer specificity to the RNA.
1 Watts, J. M. et al. Architecture and secondary structure of an entire HIV-1 RNA genome. Nature 460, 711-716, doi:10.1038/nature08237 (2009).
2 Lu, K., Heng, X. & Summers, M. F. Structural determinants and mechanism of HIV-1 genome packaging. Journal of molecular biology 410, 609-633, doi:10.1016/j.jmb.2011.04.029 (2011).
3 Rambo, R. P. & Tainer, J. A. Improving small-angle X-ray scattering data for structural analyses of the RNA world. RNA 16, 638-646, doi:10.1261/rna.1946310 (2010).
4 Sundquist, W. I. & Krausslich, H. G. HIV-1 assembly, budding, and maturation. Cold Spring Harbor perspectives in medicine 2, a006924, doi:10.1101/cshperspect.a006924 (2012).
5 Chamanian, M. et al. A cis-Acting Element in Retroviral Genomic RNA Links Gag-Pol Ribosomal Frameshifting to Selective Viral RNA Encapsidation. Cell host & microbe 13, 181-192, doi:10.1016/j.chom.2013.01.007 (2013).
6 Webb, J. A., Jones, C. P., Parent, L. J., Rouzina, I. & Musier-Forsyth, K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging. RNA, doi:10.1261/rna.038869.113 (2013).
Model for selection and packaging of [vRNA] by the first few binding Gag molecules. Non-ψ binding is characterized by Gag binding in a NC- and MA-bound conformation, but Gag binds ψ in an NC-only binding mode. The NC-only mode leaves MA free to interact with the membrane and has a kinetic advantage over complexes in which MA is bound to nucleic acids. Figure and legend are from , with minor modifications.