A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand,
in fulfilment of the requirements for the degree of
Doctor of Philosophy
Johannesburg 2014 / Human receptor CD4 is a membrane-bound glycoprotein expressed on the surface of certain
leukocytes where it plays a key role in the activation of immunostimulatory T cells. This
function is diverted by the Human Immunodeficiency Virus (HIV) envelope glycoprotein
(gp120), which uses CD4 as its primary receptor for cell entry. The requirement of CD4 for
viral entry has rationalised the development of recombinant CD4-based proteins as
competitive viral attachment inhibitors and immunotherapeutic agents. While growing
evidence suggests that redox exchange reactions involving CD4 disulphides (potentially
catalysed by cell surface-secreted oxidoreductases) play an essential role in regulating the
activity of CD4, their mechanism(s), biological utility and structural consequences that may
be applicable to the designs of novel antiviral therapies and vaccines remain incompletely
understood.
Herein, a novel recombinant CD4 protein designed to bind gp120 through a targeted
disulphide-exchange mechanism is described. This molecule contains a conservative Ser60
to Cys mutation on the CD4 domain 1 α-helix which, according to theoretical crystal
structure modelling, positions a thiol in close proximity of the gp120 V1/V2 loop disulphide
(126Cys–Cys196) resulting in the formation of an interchain disulphide bond. Experimental
evidence for this effect is provided by describing the expression, purification, refolding,
receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant
containing the S60C mutation (2dCD4-S60C). This 2dCD4-S60C binds HIV-1 gp120 with a
significantly higher affinity than wild-type protein under conditions that facilitate disulphide
exchange and this translates into a corresponding increase in the efficacy of CD4-mediated
viral entry inhibition.
To gain more insights into the importance of redox activity in the mechanism of HIV entry, a
panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala
variants, were used to show that Thioredoxin (Trx), an oxidoreductase found on the cell
surface, reduces 2dCD4 highly efficiently, catalysing the formation of conformationally
distinct monomeric 2dCD4 isomers, and a stable, disulphide-linked 2dCD4 dimer. HIV-1
gp120 was shown to be incapable of binding a fully oxidised, monomeric 2dCD4 in which
both domain 1 and 2 disulphides are intact, but binds robustly to reduced equivalents that
are the products of Trx-mediated isomerisation. This Trx-driven dimerisation of CD4, a
process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen
presentation complexes, is shown to be impaired when CD4 is bound to gp120. Finally,
preliminary, low-resolution structural analysis of individual CD4 domains 1 and 2 are
suggestive of intrinsic metastability in domain 2, and reduction of its resident allosteric
disulphide bond likely underpins the structural rearrangements in CD4 that are required for
efficient interaction with gp120.
Overall, these findings emphasise the fundamental importance of redox pathways in the
biochemical mechanism of HIV entry, and illustrate the feasibility of exploiting these for the
development of novel antiviral ligands.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/17453 |
| Date | 20 April 2015 |
| Creators | Cerutti, Nichole Michelle |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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