Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is
an accessory protein that interacts with a number of host cellular and other viral
proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell
cycle G2 arrest, modulation of gene expression, and suppression of immune
activation. The functionality of subtype C Vpr, especially South African strains, has
not been studied. The aim of this study was to describe the diversity of South African
HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr
proteins.
Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and
subtyped using phylogenetic analysis. Fragments containing natural mutations were
cloned in mammalian expression vectors. A consensus subtype C vpr gene was
constructed and site-directed mutagenesis was used to induce mutations in postions in
which no natural mutations have been described. The functionality of all constructs
was compared with the wild-type subtype B Vpr, by transfecting human 293T cell
line to investigate subcellular localization, induction of apoptosis and cell cycle G2
arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan
Low density arrays (TLDA) was also investigated.
Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4
strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved
including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The
following mutations were constructed using site-directed mutagenesis: P14I, W18C,
Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr
localized to the nucleus but the W18C mutation disturbed the nuclear localization of
Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was
lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced
cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle
G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins
induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells.
However, an artificially designed Vpr protein containing the consensus sequences of
subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C
Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of
Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated
gene expression was similar for all constructs, indicated that apoptosis was
efficiently induced through the intrinsic pathway by the mutants.
Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a
similar ability for nuclear localization and apoptosis induction. The induction of cell
cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C
Vpr proteins. The natural mutations studied in the isolates did not disturb the
functions of subtype C Vpr and in some cases even potentiated the protein to induce
apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as
defective, since enhanced functionality would be more indicative of an adaptive role.
The increased potency of the mutated Vpr proteins suggests that Vpr may increase the
pathogenicity of HIV-1 by adapting apoptotic enhancing mutations. / AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1
(MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer
en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies
onder andere die induksie van apoptose, die induksie van selsiklus G2 staking,
modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die
funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie
beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse
MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr
proteine te ondersoek
Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is
bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met
natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr
geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie,
is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde
tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir
subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die
modulering van geen uitdrukking in die induksie van apoptose is deur TLDA
ondersoek.
Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en
4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die
FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur
mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S.
Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer,
maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van
alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe
C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl
subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die
natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel
tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig
ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te
induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle
geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van
die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van
konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief
veroorsaak is deur die intrinsieke roete.
Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n
soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van
selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die
subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig
beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare
rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die
patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6702 |
| Date | 03 1900 |
| Creators | Romani, Bizhan |
| Contributors | Engelbrecht, Susan, Glashoff, Richard H., University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 242 p. : ill. (mostly col.) |
| Rights | University of Stellenbosch |
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