M.Med. Faculty of Health Sciences, University of the Witwatersrand, 2008 / Objectives: Characterization of HIV-1 from slow progressors (SP) is important to facilitate vaccine and antiviral drug development. In order to identify virus attenuations that may contribute to slower rates of disease progression, the full viral genomes from primary isolates of six slow progressing HIV-positive children were sequenced. Methods: Primary virus biological phenotypes were determined by growth in CCR5- and CXCR4-expressing U87.CD4 cell lines. Proviral DNA was isolated from co-cultured PBMCs, and the near full-length genomes and LTR regions were PCR amplified, sequenced and analysed. Predicted amino acid (aa) sequences for all the HIV-1 proteins were extensively analyzed. Results: All primary HIV-1 isolates utilized CCR5, and were determined to be HIV-1 subtype C by phylogenetic analysis. Predicted aa sequence analysis revealed open reading frames for all HIV-1 genes which encoded for proteins of the expected length, with several exceptions. For example, isolate LT5 had a 2 aa insertion in the Vpr mitochondriotoxic domain. Isolate LT21 contained an additional 5aa in the C-terminus of tat exon 2, while the integrase enzyme in isolate LT39 had an additional 3aa at the Cterminus. Rev from isolates LT45 and LT46 did not have the characteristic subtype C 16aa truncation, and in addition, had a further 3aa. In addition, altered functional domains was noted in several isolates, such as the cAMP-dependent kinase PKA phosphorylation site in Nef (LT5), a Vpr mutation involved in decreasing pro-apoptotic activity (LT42), and the Nef ExxxLL motif involved in the interaction with AP-1 and AP-2 (LT46). Conclusions: The slower HIV disease progression in these six children may be attributed to altered protein functions. For example, LT46 Nef is unable to bind AP-1 and AP-2 and therefore inactive on CD4 endocytosis. The biological relevance of these findings requires further investigation.

Thesis (M.Sc.(Med.)(Epidemiology and Biostatistics)), Faculty of Health Sciences, University of the Witwatersrand,2009 / BACKGROUND Studies suggest an association between Bacterial Vaginosis (BV) and HIV infection; however, its temporal effect has not been greatly investigated. METHODS This is a secondary data analysis of a cohort study: set out to describe the association between BV infection and HIV acquisition. There were 750 participants enrolled in the primary cohort study. The main exposure, BV, was measured from a gram stain slide prepared from a vaginal swab. The slide was read in a laboratory qualitatively and scored by Nugents scoring. A score of 7 or above was considered positive for BV. The outcome variable (HIV) was determined by dual rapid tests and confirmed in the laboratory by a third generation ELISA. Descriptive statistics was done to describe demographic characteristics and the prevalence of BV and STIs. HIV incidence rate was calculated. Kaplan Meier survival time analysis and log rank test for significance were performed. Cox regression (univariate and multivariate) was done to determine association of BV with HIV infection. RESULTS The baseline prevalence of BV was 52 %, 95 % CI; 45 – 59. There were 21 HIV seroconversions experienced of which 7 had BV results missing and were excluded in the analysis. The remaining 14 seroconversions were followed for a mean time of 0.40 of a year and accumulated follow up time at risk of 286 person years, this represented an HIV incidence rate of 4.9 per 100 person years of follow up, 95 % CI: 2.9 – 8.27. Kaplan Meier curves revealed a higher risk of HIV-1 acquisition among women who were BV positive than the women who were BV negative. A log rank test showed that the v probability of seroconversion was different among the women depending on BV status, chi-square value 3.8, p 0.05. Controlling for confounding variables, seroconversion was high, but not significant, among BV positive women, adjusted hazard ratio 3.21; 95 % CI; 0.85-12.12, p value 0.08. CONCLUSION This study suggests that BV increases HIV seroconversion risk though statistical significance was not achieved. Vaginal cleansing education, screening and treating women with BV could maintain normal vaginal flora and reduce their susceptibility to HIV.

PhD, Faculty of Health Sciences, University of the Witwatersrand / The vast potential of the RNA interference (RNAi) pathway as a new tool for the development of therapeutic modalities has been quickly realised since its discovery in 1998. RNAi effector mimics have been developed to successfully silence an array of disease-causing genetic elements. However, because of the rapidly mutating genome of viruses such as the human immunodeficiency virus (HIV), inhibition of replication cannot be sustained with single RNAi effector mimics. Instead, a combinatorial approach is required, analogous to the cocktail of drugs necessary for successful highly active antiretroviral therapy (HAART). Pioneering studies utilizing long hairpin RNAs (lhRNAs) showed that the long double-stranded RNA stem region acts as a Dicer substrate and is processed into multiple siRNA species. This intrinsic combinatorial property of lhRNAs was exploited in this thesis by attempting to incorporate three non-contiguous potent siRNA sequences within a single lhRNA stem expressed from an RNA Pol III promoter. Although significant knockdown of three independent HIV target sequences was possible, the limitations of this approach became apparent when it was observed that human Dicer does not function efficiently as a multiple turnover enzyme. The generation of siRNA products therefore occurred in a gradient, with higher levels of siRNA produced from the base of the hairpin stem and decreasing quantities generated towards the loop. Modifications to the configuration of integrated siRNA sequences within the stem region enabled augmented RNAi activity of siRNAs in the second position of the hairpin stem. This led to the notion that further manipulation of the structural design of the stem duplex may improve efficacy of up to two siRNAs. Dual-targeting anti-HIV lhRNAs encoding only two highly effective siRNAs targeted against non-contiguous sites within the tat, nef, LTR and int viral genes were therefore propagated. The spatial arrangement of two siRNA sequences was extensively characterised within dual-targeting lhRNAs by inserting up to three random base pairs at the junctions of siRNA encoding sequences and 5 bp preceding the terminal loop sequence. A universally optimal hairpin design was identified which contained a single mismatched base pair between two 19 bp + 2 nt siRNA sequences, as well as a terminal extension. Two powerful dual-targeting lhRNA species, lhRNA-tat-nef +1 and lhRNA-LTR-int +1, each capable of producing two potent anti-HIV siRNA products in equal quantities were selected for incorporation into a combinatorial RNAi system. These two effective dual-targeting lhRNAs were combined, adjacent to one another within a single RNA Pol III-expressed transcript to create a novel lhRNA-based combinatorial RNAi structure. This double lhRNA (dlhRNA) construct served as a precursor for four discrete highly functional RNAi effector sequences which were capable of simultaneously silencing four unique HIV target sites within the tat, nef, LTR and int genes. Furthermore, the ectopic expression of dlhRNAs did not elicit activation of the interferon response, nor did it cause saturation of the endogenous miRNA biogenesis pathway in vitro. In conclusion, the inherent combinatorial RNAi properties of long hairpin RNAs were evaluated and the detailed analysis is presented in this thesis. Structurally optimised dualtargeting lhRNAs subsequently formed the core components of a novel dlhRNA precursor which meets all the requirements for an effective combinatorial RNAi strategy and therefore holds great promise for mediating an effective and sustained gene therapy against HIV.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand , in fulfillment of the requirements for the degree of Master of Science in Medicine, 2013 / Sequence analysis from HIV-1 (human immunodeficiency virus type 1) subtype B and more recently subtype C infected patients has revealed that mutations in the HIV-1 protease region that confer drug resistance to boosted protease inhibitor (PIs) are rarely detected at the time of virological failure. Mutations in the HIV-1 subtype B gag-pol cleavage sites are thought to be compensatory mutations which arise as a result of PI use. This study investigated the presence of compensatory mutations in the HIV-1 subtype C gag-pol cleavage sites and matched pol genotypes from South African patients failing a boosted PI-based regimen, as compared to antiretroviral drug naïve patients. A new amplification protocol encompassing the near full-length gag, PR and partial RT was established and used to sequence the HIV-1 gag-pol cleavage sites from 23 proviral DNA samples (p24 antigen cultured peripheral blood mononuclear cells; PBMCs), and 51 patient samples (23 antiretroviral drug-naïve, 26 failing second-line lopinavir/ritonavir containing regimens), all attending the Charlotte Maxeke Johannesburg Hospital. Nucleotide sequences were aligned and codon positions S373Q, A431V, I437T/V, L449P or P453L associated with known gag-pol cleavage site mutations were analysed and compared. The pol genotypes were established using an in house assay. Antiretroviral drug resistant primary virus isolates were grown from samples from patients enrolled on the CIPRA-SA study, and propagated in coculture with PHA-activated, IL-2 stimulated PBMCs. HIV-1 gag-pol cleavage sites and pol genotypes for all primary virus isolates were established as described above. Fifty one of 74 patient samples, used to establish the in-house gag-pol cleavage site assay, were successfully amplified and sequenced. Detailed analysis of the five known gag-pol cleavage sites revealed that 5 patient samples (4 PI-exposed, 1 unknown regimen) encoded for the previously described mutations that impact on gag-pol cleavage in the absence of any major PR mutations. A further five samples from patients on the failing PI-based regimen had major PR mutations. No known mutations in the gag-pol region were identified in patients failing a first line regimen. The pol mutations described in this study were similar to the findings reported for treatment failures in South African HIV-1 subtype C infected patients. Primary virus was grown from only 25 of the 91 PBMC CIPRA samples. None of the 25 CIPRA-SA primary virus isolates had gag-pol cleavage site mutations, and only 9 harboured known RT antiretroviral drug resistant mutations. Overall, the presence of HIV-1 gag-pol cleavage site mutations may account for virological treatment failure in 5 of the South African patient samples analysed. Although the gag-pol cleavage site mutations detected in the current study are only present in a small proportion of treatment-experienced South African patients, this may increase due to more patients accessing second line PI-containing regimens. Thus, future genotyping work incorporating the analysis of the gag-pol cleavage sites in addition to the PR and RT regions is warranted. The antiretroviral drug resistant primary viruses obtained provide valuable reagents for future phenotyping studies.

An important step in the production of infectious HIV-1 particles is maturation of the virus core. This process is completed by cleavage of the capsid (CA) domain of Gag, from its precursor, CA-SP1, by the viral protease. Large deletions in stem-loop 1 (SL1) in the 5' untranslated region (UTR) of HIV-1 genomic RNA (gRNA) delay CA-SP1 processing. SL1 harbours the dimerisation initiation site (DIS) palindrome suggesting that efficient Gag processing may be linked to gRNA dimerization as shown in HIV-2. However, a dimerisation mutant with normal Gag processing was identified. Gag processing defects are hallmarks of late domain mutants, and SL1 mutation was found to result in reduced virus release. HIV-1 hijacks the host's endosomal complexes required for transport (ESCRT) pathway to enable budding. An ESCRT-associated protein, ALIX, is known to be capable of binding to the nucleocapsid (NC) domain of Gag using lipids or RNA as a 'bridge' in vitro. It was hypothesised that SL1 mutation disrupts an RNA-dependent interaction that occurs during virus assembly. Consistent with this, an intact SL1 was found to be required for efficient ALIX function. Increasing the abundance of gRNA in the cell by expressing it in trans accelerated CA-SP1 processing in a manner that required ALIX's binding motif in p6. Gag processing could also be accelerated by introducing previously identified compensatory mutations into the SP1 and NC domains of Gag, in a manner reminiscent of the actions of maturation inhibitor resistance mutations. The effects of the compensatory mutations were also dependent on intact late domain motifs. These data suggest that gRNA is involved in regulating virus budding and maturation through interaction with ALIX. A model is proposed whereby the packaging signal (psi) region of gRNA acts as a bridge between Gag and ALIX, acting as a checkpoint mechanism to promote Gag processing and optimise release of virions that have successfully packaged gRNA.

La vitesse de réplication du HIV-1(Human Immunodeficiency Virus type 1), qui semble corrélée de manière directe à la vitesse de progression de la maladie vers le stade SIDA, est essentiellement contrôlée au niveau transcriptionnel. La transcription du HIV-1 est régulée par la structure chromatinienne, des éléments agissant en cis localisés dans les LTRs, des facteurs de transcription agissant en trans et par la protéine virale trans-activatrice Tat (revu dans Quivy et al. 2007, Bisgrove et al. 2005, Rohr et al. 2003, Rabson and Graves 1997). En plus de l’enhancer localisé dans le LTR5’ du HIV-1, un enhancer intragénique, localisé dans le gène pol du HIV-1, inductible par le phorbol 12-myristate 13-acétate (PMA) a été identifié. La localisation progressive de l’activité enhancer a permis de définir deux domaines distincts et indépendants dans cet enhancer intragénique : les fragments 5103 et 5105 localisés respectivement dans la partie centrale du gène pol et dans une région couvrant la fin du gène pol, le gène vif, le gène vpr et le premier exon codant des gènes tat et rev (Verdin et al. 1990). Les fragments 5103 et 5105 se comportent tous deux comme des enhancers inductibles par le PMA lorsqu’ils sont clonés en amont du promoteur de la thymidine kinase dans un vecteur rapporteur en cellules HeLa. Notre laboratoire a précédemment identifié trois sites de liaison pour les facteurs de transcription AP-1 dans le fragment 5103 (Van Lint et al., 1991). Au cours de notre thèse, nous avons poursuivi la caractérisation de ces sites de liaison AP-1 et avons montré que les facteurs c Fos, JunB et JunD interagissent in vitro avec ces motifs. Pour chaque site, nous avons identifié des mutations qui abolissent la liaison des facteurs AP-1 sans altérer la séquence en acides aminés sous-jacente de la transcriptase inverse. Par des expériences de transfection transitoire, nous avons démontré que les sites AP 1 intragéniques sont entièrement responsables de l’activité enhancer PMA-dépendante du fragment 5103. De plus, l’activité PMA-inductible du fragment 5103 est inhibée par le mutant dominant négatif A-Fos à condition que les sites ne soient pas mutés. A l’inverse, l’expression ectopique de dimères forcés AP-1 affecte positivement l’activité enhancer du fragment 5103. Enfin, nous avons étudié le rôle biologique des sites AP-1 intragéniques dans la réplication virale et avons montré que ces sites contribuent positivement à l’infectivité du virus. Durant la seconde partie de notre thèse, nous avons entamé la caractérisation physique et fonctionnelle du fragment 5105. Nos résultats de transfection transitoire montrent que l’activité PMA inductible du fragment 5105 est localisée dans le dernier tiers de ce dernier : le sous fragment 5105.3. L’analyse bioinformatique de cette région a permis de mettre en évidence un site de liaison pour les facteurs AP-1 in vitro. Des mutations ponctuelles permettent d’abolir la liaison des facteurs à leur site mais altèrent la séquence en acides aminés sous-jacente codant pour les protéines Tat et Rev. Nous avons montré que ce site est impliqué dans l’activité transcriptionnelle de ce fragment. L’expression ectopique du mutant dominant négatif A-Fos inhibe l’activité transcriptionnelle PMA-inductible du fragment 5105. Une analyse bioinformatique plus large nous a ensuite permis d’identifier in vitro, par retard de migration sur gel, 5 sites de liaison pour le facteur YY1 et 2 sites de liaison pour le facteur PU.1 dont les implications pour le virus restent encore à déterminer.

Protein transduction domains (PTDs) are short peptide sequences that can transport wide varieties of cargo across cell membranes. This study assessed the transduction ability of fusion proteins containing optimised variants of the PTD from HIV-1 transactivator of transcription (Tat). Uptake of Tat-PTDs was determined by fluorescent microscopy using the fluorescent protein Venus as a tag, and also by using fusion proteins containing caspase-7 and RhoA bound to Tat-PTD. Upon entering the cytosol the latter two induce apoptosis and the formation of cytoplasmic extensions, morphological changes easily observed by microscopy. It was found that PTDs with two, three or four sequential Tat-PTD domains could bind to the surface of two of the five cell lines tested. Fluorescent microscopy, however, indicated that the fluorescent constructs remained on the cell surface. As well, PTDs bound to caspase-7 or RhoA did not induce any visible morphological changes in the cells.

Protein transduction domains (PTDs) are short peptide sequences that can transport wide varieties of cargo across cell membranes. This study assessed the transduction ability of fusion proteins containing optimised variants of the PTD from HIV-1 transactivator of transcription (Tat). Uptake of Tat-PTDs was determined by fluorescent microscopy using the fluorescent protein Venus as a tag, and also by using fusion proteins containing caspase-7 and RhoA bound to Tat-PTD. Upon entering the cytosol the latter two induce apoptosis and the formation of cytoplasmic extensions, morphological changes easily observed by microscopy. It was found that PTDs with two, three or four sequential Tat-PTD domains could bind to the surface of two of the five cell lines tested. Fluorescent microscopy, however, indicated that the fluorescent constructs remained on the cell surface. As well, PTDs bound to caspase-7 or RhoA did not induce any visible morphological changes in the cells.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 77-90).

Inducible LAK cell responses were studied in HIV-seropositive individuals lacking clinical symptoms, and overt AIDS patients. Inducible LAK cell responses have been operationally defined as, non-MHC-restricted and antigen-nonspecific cytotoxic activity observed following IL-2 stimulation. HIV-seropositive asymptomatic individuals exhibited an enhanced LAK cell response against HIV-infected targets while lysis of uninfected targets remained at control levels. LAK activity of AIDS patients however, was significantly diminished when compared to healthy controls. Immunomagnetic negative selection depletion experiments indicated that LAK cell activity is mediated primarily by CD56-expressing lymphocytes, both at the progenitor and effector cell level. Of interest, in HIV-seropositive asymptomatic individuals we observed the emergence of a second CD8-expressing cytotoxic population that mediates IL-2-induced non-MHC-restricted and antigen-nonspecific cytotoxicity. Overall we demonstrated that CD56-expressing LAK cells of HIV-seropositive patients exhibited a decreased ability to mediate cytotoxicity on a per cell basis against a panel of different targets. In vivo, this inhibition may be amplified by decreases in absolute numbers of CD56-expressing lymphocytes per ml of blood. HIV-infection therefore results in dramatic changes on the number, function and phenotype of the effector cells mediating IL-2 inducible LAK cell responses.