Human Immunodeficiency Virus type I genome consists of two identical RNA molecules that are non-covalently linked to form a dimer. HIV-1 immature and mature genomic RNA (gRNA) dimers were found in protease defective (PR -) and wild type virions, respectively, and the 5'untranslated region (5' UTR) was shown to play key roles during the genome dimerization process; but the dimerization mechanism still remains to be clarified My research project is to characterize the dimerization process and the role of 5' UTR in genome dimerization in virions produced by tissue culture cells. I'll firstly show the dimer maturation processes of HIV-1 gRNA isolated from newly released to grown-up (≥10h old) wild type, PR-, and SL1 defective (DeltaIDS) virions respectively. The results showed that HIV-1 gRNA dimer maturation process was protease-dependent and involved multiple steps: from low to high dimerization level and dimer thermostability, and from low dimer mobility to intermediate and high mobility. PR- virions did not freeze gRNA conformation in the primordial nascent state and gRNA changed from monomeric in newly released virions to half dimeric in grown-up virions, which showed that genome was packaged in the form of monomeric RNA or fragile dimers, more thermolabile than immature dimers in grown-up PR- virions. DeltaDIS inhibited gRNA dimerization by about 50% in newly released virions, though grown-up DeltaDIS gRNA was fully dimeric, which indicated that the DIS played the initiation role in gRNA dimerization in HIV-1 virions. The gRNA dimerization rate in PR- or DeltaDIS virions was much slower than that in wild type virions. These results show for the first time the whole process of dimer maturation after virion release, the gRNA conformation rearrangement in PR- virions, and the initiation role of the DIS in HIV-1 virions. Next, I'll provide a rather systematic search for the contribution of different regions in 5' UTR to HIV-1 gRNA dimerization by studying selected mutations singly or together with defective SL1. The results showed that the 5'trans-activation response element (5'TAR) was directly involved in gRNA dimerization, and a long distance base-pairing interaction between a sequence in U5 region (nts105-1l5) and another around the initiation codon of the gag gene (nts334-344) was structurally contributive to gRNA dimerization. Deletions of sequences around the 3'end of Primer Binding Site (PBS) stem-loop moderately decreased gRNA dimerization level. Other sequences in 5' UTR except DIS/SL1, which was previously known to play important roles, didn't show any systematic role. Here the results suggested that the absence of inhibition on gRNA dimerization level with defective DIS might be the compensation of the direct role of 5'TAR; and wild type-like dimerization level of DeltaTAR must be the direct contribution of the DIS.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111918 |
| Date | January 2008 |
| Creators | Song, Rujun. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002827290, proquestno: AAINR66693, Theses scanned by UMI/ProQuest. |
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