Identification and structural characterization of a high-affinity nucleocapsid protein binding site within the human immunodeficiency virus type 2 leader RNA: Implications for genome recognition

Title	Identification and structural characterization of a high-affinity nucleocapsid protein binding site within the human immunodeficiency virus type 2 leader RNA: Implications for genome recognition
Author	Johnson, Barry
Advisors	Summers, Michael F.
Program	Biology, Molecular and Cell
UMBC Department	Molecular and Cell Biology
Document Type	dissertation
Sponsors	University of Maryland, Baltimore County (UMBC)
Keywords	retrovirus; HIV; nucleocapsid; RNA; NMR spectroscopy; Chemistry, Biochemistry (0487)
Date Issued	2008-02-05; *
Abstract	Genome packaging is a key step in retrovirus replication. Two copies of the virus' RNA are specifically selected and packaged into an assembling virion. The nucleocapsid (NC) domain of the Gag polyprotein and the packaging signal (?-site) region of the genomic RNA are critical to this process. Different packaging mechanisms have been proposed for HIV-1 and HIV-2. In HIV-1, the ?-site is downstream of the major splice donor (SD), and any splicing removes both NC binding sites. The proposed HIV-2 ?-site is upstream of the SD. A "co-translational packaging" hypothesis was presented explaining this contradiction. In this mechanism, NC binds the full-length RNA from which it was translated. Other studies indicated the region between the SD and the Gag initiation codon is critical for NC binding and genome packaging. This finding supports the hypothesis that HIV-1 and HIV-2 package their genomes in a similar manner. However, the minimal NC binding domain(s) in HIV-2 had not been determined. The work presented here identifies such an NC binding element downstream of the SD in HIV-2 and shows the proposed packaging signal upstream of the SD binds NC with a dissociation constant ~6-fold greater than the downstream site. In addition, other structural elements in this region bind NC with even lower affinity. The primary HIV-2 NC binding site is an unstructured linker RNA sequence, whereas the two primary binding sites in HIV-1 are ordered tetraloops. This difference in RNA structure led to the hypothesis that the NC proteins of HIV-1 and -2 behave differently. The three-dimensional structure of HIV-2 NC was determined by NMR spectroscopy. This protein appears to be more rigid than its HIV-1 counterpart. An oligonucleotide corresponding to the ?3-AUG linker is unstructured in solution, but adopts an ordered conformation upon binding to NC. The different levels of order of both the proteins and the RNAs in these two viruses could explain previous work showing a non-reciprocal cross-packaging relationship when one virus' NC was replaced with that of the other virus. In this way, understanding the interactions involved in genome packaging at the molecular level could lead to the development of more efficient and safer vectors for gene delivery trials.
Identifier	1106
Format	application:pdf
Language	en
Collection	UMBC Theses and Dissertations
Rights Statement	This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu.

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Identification and structural characterization of a high-affinity nucleocapsid protein binding site within the human immunodeficiency virus type 2 leader RNA: Implications for genome recognition

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