ETD Collection

Permanent URI for this collectionhttps://wiredspace.wits.ac.za/handle/10539/104


Please note: Digitised content is made available at the best possible quality range, taking into consideration file size and the condition of the original item. These restrictions may sometimes affect the quality of the final published item. For queries regarding content of ETD collection please contact IR specialists by email : IR specialists or Tel : 011 717 4652 / 1954

Follow the link below for important information about Electronic Theses and Dissertations (ETD)

Library Guide about ETD

Browse

Filters

20101

Settings

Author: search.filters.author.Bell, Catherine MacdonaldSubject: search.filters.subject.HIV-1

Search Results

Now showing 1 - 1 of 1
  • No Thumbnail Available
    Item
    Differential timing of translocation of HIV-1 subtype B and C Vpu to the ER/Golgi an plasma membrane compartments
    (2010-04-19T12:36:01Z) Bell, Catherine Macdonald
    The HIV-1 Vpu protein functions largely to target CD4 molecules for proteasomal degradation, and to enhance virion release. The subcellular localisation of Vpu is related to these functions. Previous studies showed subtype B Vpu localisation at the ER/Golgi complex, while subtype C Vpu localised to the plasma membrane (PM) at 48 hours post-transfection. To determine if subtype C Vpu can localize to the ER/Golgi, we evaluated the cellular localisation of Vpu from two South African subtype C isolates as compared to subtype B Vpu, over time. Codon optimized vpu genes from subtype C isolates FV5 and FV15 (which have a six and two amino acid insert in the N-terminal domain, respectively) and a representative subtype B vpu were TA cloned into the pcDNA6.2/C-emGFP expression vector. The three VpuemGFP recombinant plasmids were cotransfected with pDsRed-ER, pDsRed-Golgi, or pDsRed-Mem into HEK 293T cells, and observed at 24, 48, and 60 hours posttransfection under a confocal microscope to confirm the presence of Vpu at different subcellular compartments. Cotransfection and microscopy conditions were methodically optimised. At 24 hours post-transfection, the subtype C FV5 Vpu had ER/Golgi localisation, but none at the PM. The subtype C FV15 Vpu had weaker ER/Golgi localisation and no PM localisation. In contrast, the subtype B Vpu had strong PM localisation. At 48 hours, FV5 and FV15 Vpu showed PM localisation, while subtype B Vpu was clearly localised at the ER/Golgi. At 60 hours, FV5 Vpu was observed at the PM, whereas FV15 and subtype B Vpu showed ER/Golgi localisation. These findings illustrate the efficient translocation of Vpu between different cellular compartments and for the first time, the difference in timing between subtype B and C Vpu, as well as íntrasubtype differences. This difference in shuttling suggests implications for the timing of viral assembly and release. Further investigations may clarify the impact of this timing on the difference in disease pathogenesis noted between infections with the different subtypes.