Human immunodeficiency virus type 1 (HIV-1) genome consists of two identical RNAs that appear noncovalently linked near their 5' ends. The 5' untranslated region is called leader region. The 3' part of leader, i.e. nucleotides U200 to G335 in HIV-1 genomic RNA, between the primer binding site and the gag gene, can fold into 3 stem-loops: the kissing-loop domain (KLD) or stem-loop 1 (SL1), the 5' splicing junction hairpin (SD) or SL2 and SL3. The KLD, from nucleotide (nt) 243 to 277, forms a stem-loop (kissing loop hairpin) seated on top of a small stem bulge (stem B and loop B). The kissing-loop hairpin, or dimerization initiation site (DIS) hairpin consists of stem C and loop C. Loop C contains the autocomplementary sequence (ACS) GCGCGC262 or GUGCAC262, also called DIS. / In the kissing-loop model of HIV-1 genome dimerization, HIV-1 RNA dimerization is initiated by base pairing between the ACS of one RNA monomer and that of an adjacent monomer. / To understand the role of the ACS in HIV-1 replication and HIV-1 genomic RNA dimerization, we replaced the central CGCG261 (or tetramer) of the HIV-1 Lai ACS by other tetramers. Genomic RNAs containing the UUAA tetramer (non-HIV-1 tetramer) were half dimeric, but UUAA genome packaging was unaffected. This was the first evidence that genomic RNA dimerization and packaging can be dissociated (Chapter 2). Destroying stem-loop C reduced genomic RNA dimerzation by ~50%, proviral DNA synthesis by ~85%, and reduced viral infectivity by ~3 logarithmic units. Destroying stem-loop B had similar effects on genome dimerization, reverse transcription, and viral infectivity. We also observed that mutations in stem-loop B and in the DIS hairpin were "non additive" (Chapter 2). / The existence of stem-loop C is supported by phylogenetic evidence, while that of stem-loop B is not, namely, its sequence is completely conserved. We investigated the role of stem B and loop B nucleotides in viral replication, and genomic RNA dimerization. The putative CUCG246/CGAG277 duplex was replaced by 9 alternative complementary sequences, 4 likely to base pair in long (~500 nts) RNAs, as assessed by the algorithm mfold. Among the 4 sequences, 3 preserved genome dimerzation, 1 did not significantly inhibit it, and 2 preserved viral replication. We also asked if 9 deletions or nucleotide substitutions within nucleotide 200 to 242 and/or 282 to 335 could influence genome dimerization. Delta200--226 and Delta236--242 genomic RNAs dimerized relatively poorly despite having neutral or positive influences on stem B, loop B and klh folding (Chapter 3). / Mutations within the Matrix, Capsid, p2 and nucleocapsid genes suppress several functional defects caused by KLD destruction. We tested the effect of these suppressor mutations on genome dimerization and infectiousness of viruses bearing moderate to crippling KLD mutations. Our conclusion is that these suppressor mutations can restore genomic RNA dimerization when DIS is weakened, but not when DIS is denatured or the KLD is destroyed (Chapter 4).
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84434 |
| Date | January 2002 |
| Creators | Shen, Ni, 1969- |
| Contributors | Laughrea, Michael (advisor) |
| 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: 001975149, proquestno: AAINQ88578, Theses scanned by UMI/ProQuest. |
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