The characterisation and expression of HIV-1 subtype C gag

Sampson, Candice Corene (2002)

Thesis (MScMedSc) -- University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL responses are important in controlling viral load during acute infection and asymptomatic stages of the infection. Currently, only one complete South African HIV-1 subtype C gag sequence has been published. The first aim of this study was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be used as a set of reference sequences in the design of a South African HIV-1 subtype C vaccine. Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998 and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned into mammalian expression vectors and sequenced. Restriction digest analyses as well as phylogenetic analyses were performed on the sequencing data. Previously published mutational analyses and CTL epitopes were compared to the predicted amino acid sequences of the gag clones. Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C isolates as well as one complete sequence of an HIV-1 subtype B isolate were compiled. Subtyping by restriction fragment length polymorphism (RFLP) would have correctly identified 14 of the 15 subtype C isolates as subtype C and one as unidentifiable. The subtype B isolate would have also been correctly identified. Phylogenetic analyses showed that our subtype C isolates clustered with reference subtype C strains from various countries, including Botswana, India, Israel, Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype C cluster. The diversity between our isolates was comparable to the diversity seen between all the HIV-1 subtype C strains. Comparisons of previously published mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the sequence. A second aim was to establish transfection and Western Blot techniques in our laboratory for use in future studies. An in vitro transcription! translation assay was performed on the gag clones and the protein producing clones were used to transfect mammalian cells using electroporation. A Western blot was then used to screen for Gag protein expression in the transfected cell Iysates. The in vitro transcription! translation assay showed that seven of the 23 clones could produce a protein of -55 kDa in size. Four out of the seven of these clones gave a weak expression of a-55 kDa protein after transfection in a mammalian cell line. Since the completion of the experimental work of this study, other cloned HIV-1 genes have successfully been transfected into mammalian cells using the electroporation technique and the proteins produced were screened for by Western blot. To conclude with; the native form of the gag gene does not elicit strong expression of the protein, but studies have shown that expression can be improved by sequence-modification of the gag nucleotide sequence. Due to the conservation of gag, the sequence of any subtype C strain can be used for the development of a Southern African vaccine.

AFRIKAANSE OPSOMMING: Die HIV-1 gag geen kodeer vir een van die hoof strukturele proterene en bevat verskeie sitotoksiese T-limfosiet epitope. Gag spesifieke sellulere immuun respons is belangrik vir die beheer van virale lading tydens akute infeksies en tydens asimptomatiese fases van die infeksie. Tans is slegs een volledige Suid Afrikaanse HIV-1 subtipe C nuklerensuur volgorde gepubliseer. Die eerste doel van hierdie studie was om die volledige gag geen van 15 HIV-1 subtipe C isolate te karakteriseer, om gebruik te word as In stel verwysings nukleiensuur volgordes, vir die ontwerp van In Suid Afrikaanse HIV-1 subtipe C entstof. Die 15 HIV-1 subtipe C isolate wat vir hierdie studie geselekteer is, is tydens 1998 en 1999 ge·lsoleer vanaf HIV-1 positiewe pasiente wat die Infeksiesiekte Kliniek, Tygerberg Hospitaal bygewoon het. Die gag geen van hierdie isolate is geamplifiseer deur PKR, gekloneer in soogdier ekspressie vektore en die nukleiensuur volgorde is bepaal. Die nuklerensuur volgorde is gebruik in restriksie ensiem analises asook filogenetiese analises. Reeds gepubliseerde mutasie analises en limfosiet epitope is met die voorspelde aminosuur volgorde van die gag klone vergelyk. Die nukleiensuur volgordes van die 23 volledige gag gene wat die 15 HIV-1 subtipe C isolate verteenwoordig, asook een volledige nukleiensuur volgorde van een HIV-1 subtipe B isolaat, is saamgestel. Subtipering deur middel van restriksie fragment lengte polimorfisme (RFLP) sou 14 uit die 15 subtipe C isolate korrek qerdentifiseer het, maar sou een nie kon identifiseer nie. RFLP sou ook die subtipe B isolaat korrek qerdentifiseer het. Filogenetiese analises het gewys dat ons subtipe C isolate met die verwysings subtipe C stamme van verskeie lande, insluitend Botswana, lndie, Israel, Tanzania en Zambie groepeer. Stamme van Ethiopie en Brasilie het In aparte subtipe C groep gevorm. Die diversiteit tussen ons isolate was vergelykbaar met die diversiteit tussen al die subtipe C stamme. Vergelykings van gepubliseerde mutasie analises en limfosiet epitope met die voorspelde aminosuur volgorde van die gag klone, het konservasie in meeste van die klone, deur die hele nukleiensuur volgorde, getoon. Die tweede doel was om die metodes van transfeksie en Westerse klad in ons laboratorium tot stand te bring. In vitro transkripsie/ translasie toetse is gedoen op die gag klone en die proteten produserende klone is gebruik om soogdierselle te transfekteer deur gebruik te maak van elektroporasie. In Westerse klad is toe gebruik om vir Gag proterenuitdrukkinq in die sellisate te toets. Die in vitro transkripsie/ translasie toets het getoon dat sewe uit 23 klone, In proteren van -55 kDa kon produseer. Vier uit die sewe van hierdie klone het In -55 kDa proteren swak uitgedruk na transfektering van soogdier selle. Sedert die voltooiing van die eksperimentele werk van hierdie stud ie, is ander gekloneerde HIV-1 gene suksesvol in soogdierselle getransfekteer met die gebruik van elektroporasie en die proterene is met In Westerse klad aangetoon. Ten slotte: die natuurlike vorm van die gag geen ontlok nie In sterk ekspressie van die proteren nie, maar ander studies het wei aangetoon dat die ekspressie verbeter kan word met modifikasie van die gag nukleiensuur volgorde. As gevolg van die konservasie van gag, kan die nuklerensuur volgorde van enige subtipe C stam gebruik word vir die ontwikkeling van In Suider Afrikaanse entstof.

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