MSc (Med) Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / In recent years there has been much progress in understanding and defining the key
protein structure-function relationships that mediate Human Immunodeficiency Virus
(HIV-1) entry into host CD4+ cells. This process involves fusion of the virus and host
cell membranes, following engagement of corresponding viral (gp120) and target
(CD4) receptor proteins. Binding of gp120 to CD4 triggers extensive conformational
changes in gp120, exposing binding sites for the co-receptor proteins (CCR5 or
CXCR4), and facilitating insertion of gp41, the viral fusion protein, into the target cell
membrane. Following insertion of gp41, oligomerisation of fusogenic domains on
gp41 is thought to drive the juxtaposition of the virus and host cell and fusion of their
membranes. Recent reports suggest that detergent-resistant membrane domains,
known as lipid rafts, play a crucial role in orientating the receptor molecules during
this step of HIV-1 infection. Lipid rafts are typically rich in cholesterol, sphingolipids
and GPI-anchored proteins, and are biophysically distinct from the glycerophosolipid
bilayer, which constitutes the bulk of mammalian cell membranes. The role of lipid
rafts in virus entry, however, is still controversial, and further experimentation is
needed to define their importance in this regard. To provide insight into the role of
lipid rafts during HIV-1 entry, we evaluated the natural distribution of the host receptor
proteins in HIV-1 target cells (U87.CD4.CCR5/CXCR4). CD4 was detected in membrane
samples fractionated by sucrose density gradient centrifugation using
immunoblotting techniques, while CCR5 and CXCR4 were detected on whole cells by
fluorescence microscopy. We then used a primary CCR5-utilising subtype C HIV-1
isolate (FV5) to characterise dynamic changes in the distribution of these receptors
and gp41 during viral entry in real-time. Viral fusion assays were set up by inoculating
v
target cells with FV5 at 23 ºC, a temperature that allows HIV-1 attachment, but is nonpermissive
for advancement of the fusion reaction. This prefusogenic form of the
virus-host receptor complex is defined as the temperature-arrested state (TAS). We
found that, under normal, uninfected conditions, CD4, CCR5 and CXCR4 are
distributed throughout both raft and non-raft microdomains on the U87 cell surface,
and there is little evidence for any significant redistribution of these receptors into
lipid rafts during the HIV-mediated fusion reaction. Interestingly, we observed a
change in the structure of CD4 during the fusion process, which could describe a
functionally important event in HIV-1 entry, or be related to compromises in the
integrity of the virally-infected membranes. Moreover, we discovered that gp41 is
capable of membrane insertion and oligomerisation at TAS, in contrast to previous
reports that suggest the fusion peptide is not capable of breaching the membrane at
this temperature. Our results provide valuable novel insights into the HIV-1 subtype C
entry process, and the involvement of lipid rafts in this stage of the viral lifecycle, that
may be relevant to novel therapy and immunogen design.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/8835 |
| Date | 18 October 2010 |
| Creators | Jamieson, Emma |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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