Browsing by Author "Madlala, Paradise Zamokuhle."

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Association of genetic polymorphisms in select HIV-1 replication cofactors with susceptibility to HIV-1 infection and disease progression.
(2011) Madlala, Paradise Zamokuhle.; Ndung'u, Peter Thumbi.; Kormuth, Emil.
Objective.Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and disease progression. This heterogeneity is attributed to the interplay between the environment, viral diversity, immune response and host genetics. This study focused on host genetics. We studied the association of single nucleotide polymorphisms (SNPs) in peptidyl prolyl isomerase A (PPIA), transportin 3 (TNPO3) and PC4 or SFRS1 interacting protein 1 (PSIP1) genes with HIV-1 infection and disease progression. These genes code for Cyclophilin A (CypA), Transportin-SR2 (TRN-SR2) and Lens epithelium derived growth factor/p75 (LEDGF/p75) proteins respectively, which are all validated HIV replication cofactors in vitro. Methods. One SNP A1650G in the PPIA gene was genotyped in 168 HIV-1 negative and 47 acutely infected individuals using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 6 intronic and 2 exonic haplotype tagging (ht) SNPs (rs13242262; rs2305325; rs11768572; rs1154330; rs35060568; rs8043; rs6957529; rs10229001) in the TNPO3 gene, 4 intronic ht SNPs (rs2277191, rs1033056, rs12339417 and rs10283923) and 1 exonic SNP (rs61744944, Q472L) in the PSIP1 gene were genotyped in 195 HIV-1 negative and 52 acutely infected individuals using TaqMan assays. The rs1154330, rs2277191, rs12339417 and rs61744944 were further genotyped in 403 chronically infected individuals. CypA and LEDGF/p75 messenger RNA (mRNA) expression levels in peripheral blood mononuclear cells (PBMCs) were quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The impact of the Q472L mutation on the interaction of LEDGF/p75 with HIV-1 integrase (IN) was measured by AlphaScreen. Results. The minor allele (G) of SNP A1650G (1650G) in the promoter region of PPIA was significantly associated with higher viral load (p<0.01), lower CD4+ T cell counts (p<0.01) and showed a possible association with rapid CD4+ T cell decline (p=0.05). The 1650G was further associated with higher CypA expression post HIV-1 infection. The minor allele (G) of rs1154330 in the intron region of TNPO3 was associated with faster HIV-1 acquisition (p<0.01), lower CD4+ T cell counts, higher viral load during primary infection (p<0.05) and rapid CD4+ T cells decline (p<0.01). The minor allele (A) of rs2277191 (rs2277191A) in the intron region of PSIP1 was more frequent among seropositives (p=0.06). Among individuals followed longitudinally, rs2277191A was associated with higher likelihood of HIV-1 acquisition (p=0.08) and rapid CD4+T cell decline (p=0.04) in the recently infected (primary infection) cohort. In contrast, the minor allele (C) of rs12339417 (rs12339417C) also in the intron region of PSIP1 was associated with higher CD4+ T cell counts during primary infection. The rs12339417C was also associated with slower rate of CD4+ T cell decline (p=0.02) and lower mRNA levels of LEDGF/p75 (p<0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 compared to nonseroconverters (p<0.01) and these levels decreased after HIV-1 infection (p=0.02). The Q472L mutation showed approximately 2-fold decrease in the association constant (Kd), suggesting stronger binding to HIV-1 integrase. Our findings demonstrate, for the first time, that genetic polymorphisms in the TNPO3 and PSIP1 genes may be associated with susceptibility to HIV-1 infection and the disease progression. These data provide in vivo evidence that TRN-SR2 and LEDGF/p75 are important host cofactors for HIV-1 replication. This is also the first study to show the association of genetic polymorphisms in the PPIA gene with disease outcome in a population (South African) with high burden of HIV-1 infection. Conclusions. Genetic variation in HIV-1 replication cofactors may be associated with disease outcome in a South African population. These data strongly support the role of these HIV replication cofactors in disease pathogenesis in vivo and suggest that these factors are possible targets for therapeutic interventions. However, these data will need to be replicated in larger cohorts to confirm the effect of these genetic variants. Further studies on how to target these factors in antiviral strategies are needed.
Association of polymorphisms in the LEDGF/p75 gene (PSIP1) with susceptibility to HIV-1 infection and disease progression.
(Lippincott Williams & Wilkins., 2011) Madlala, Paradise Zamokuhle.; Gijsbers, Rik.; Christ, Frauke.; Hombrouck, Anneleen.; Werner, Lise.; Mlisana, Koleka Patience.; An, Ping.; Abdool Karim, Salim Safurdeen.; Winkler, Cheryl Ann.; Debyser, Zeger.; Ndung'u, Peter Thumbi.
Objective: LEDGF/p75, encoded by the PSIP1 gene, interacts with HIV-1 integrase and targets HIV-1 integration into active genes. We investigated the influence of polymorphisms in PSIP1 on HIV-1 acquisition and disease progression in black South Africans. Methods: Integrase binding domain of LEDGF/p75 was sequenced in 126 participants. Four haplotype tagging SNPs rs2277191, rs1033056, rs12339417 and rs10283923 referred to as SNP1, SNP2, SNP3 and SNP4, respectively, and one exonic SNP rs61744944 (SNP5, Q472L) were genotyped in 195 HIV-1 seronegative, 52 primary and 403 chronically infected individuals using TaqMan assays. LEDGF/p75 expression was quantified by real-time RT-PCR. The impact of Q472L mutation on the interaction with HIV_1 IN was measured by AlphaScreen. Results: rs2277191 (SNP1) A was more frequent among seropositives (P=0.06, Fisher’s exact test). Among individuals followed longitudinally SNP1A trended towards association with higher likelihood of HIV-1 acquisition [relative hazard (RH)=2.21, P=0.08; Cox model] and it was also associated with rapid disease progression (RH=5.98, P=0.04; Cox model) in the recently infected (primary infection) cohort. rs12339417 (SNP3)C was associated with slower decline of CD4+ T cells (P=0.02) and lower messenger RNA (mRNA) levels of LEDGF/p75 (P<0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 (P<0.01) and these levels decreased after HIV-1 infection (P=0.02). Conclusions: Genetic variants of PSIP1 may affect HIV-1 outcomes. Further studies are needed to confirm the effect of genetic variation of PSIP1 on HIV-1 pathogenesis in different cohorts.
Association of polymorphisms in the regulatory region of the cyclophilin A gene (PPIA) with gene expression and HIV/AIDS disease progression.
(Wolters Kluwer Health., 2016) Madlala, Paradise Zamokuhle.; Singh, Ravesh.; An, Ping.; Werner, Lise.; Mlisana, Koleka Patience.; Abdool Karim, Salim Safurdeen.; Winkler, Cheryl Ann.; Ndung'u, Peter Thumbi.
Abstract available in PDF file.
Central nervous system (CNS) derived human immunodeficiency virus type 1 (HIV-1) subtype C long terminal repeat (LTR) genetic and functional variation mediates high viral load in this compartment of tuberculous meningitis (TBM) co-infected patients.
(2023) Ntshangase, Wenzile Senorita.; Madlala, Paradise Zamokuhle.
Background: Human immunodeficiency virus type 1 (HIV-1) ribonucleic acid (RNA) is characteristically lower in the central nervous system (CNS) than in plasma of antiretroviral treatment naïve patients. Paradoxically, there is higher HIV-1 viral load in the cerebral spinal fluid (CSF) than plasma of treatment naïve patients co-infected with tuberculous meningitis (TBM). The mechanisms that govern high viral replication in the CNS of TBM co-infected antiretroviral therapy naïve patients remain to be determined. Methodology: The study population comprised of 17 TBM and 3 non-TBM participants selected from an HIV-1 positive and TBM co-infected cohort. The HIV-1 viral RNA was reversed transcribed into complementary deoxyribonucleic acid (cDNA) thus the U3/R region of 3’ long terminal repeat (LTR) was amplified from CSF and plasma RNA by KAPA HiFi HotStart PCR Kits (ThermoFisher Scientific, Invitrogen™, USA). The patients CSF and plasma derived LTR were subsequently cloned into a pGL3 plasmid and further transfected in Jurkat and Astrocyte cell lines to assess the LTR transcriptional activity using Bright-Glo™ Luciferase Assay System (Promega, Madison, WI, USA). Results: CSF derived LTR had a significantly (p<0.0001) higher basal and Tat induced transcriptional activity compared to plasma derived LTR in Astrocyte (SVG) cell line. Similarly, CSF derived LTR had significantly higher (p=0.0024) Tat induced transcriptional activity compared to plasma derived LTR in Jurkat cell lines. LTR sequences containing an Adenine (A) at position 5 of the Sp1III binding site were associated with significantly high basal (p<0.0001) and Tat induced (p=0.0002) transcriptional activity compared to the LTR sequences containing a Thymine (T) at the same position when it was assessed in SVG cell. A similar case was observed in Jurkat cell lines. Consistently, CSF LTR sequences containing an A at position 5 of the Sp1III transcription binding site were associated with significantly higher HIV-1 viral load compared to LTR sequences containing a T at the same position (p=0.0093). Conclusion: Our data clearly show that CSF derived LTR from TBM co-infected individuals exhibit significantly higher transcriptional. Particularly, sequences containing the A5T mutation are significantly associated with higher LTR transcriptional activity and viral load.
Characterization of streptococcal infections in KwaZulu-Natal Durban by random amplified polymorphic DNA anaylsis and DNA macrorestriction analysis.
(2004) Madlala, Paradise Zamokuhle.; Beukes, Mervyn.
A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal.
Effect of HIV-1 subtype C Transactivator of transcription (Tat) A21P variant on TAR binding ability, nuclear levels of active positive transcription elongation factor b (P-TEFb) and viral latency.
(2023) Mkhize, Zakithi Zinhle.; Madlala, Paradise Zamokuhle.
The HIV-1 Transactivator of transcription (Tat) enhances the ability of the viral promoter 5’ long terminal repeat (LTR) to drive viral gene transcription and is important for HIV-1 pathogenesis. Tat binds to the transactivator RNA (TAR) element of the 5’LTR and subsequently recruits the host positive transcription elongation factor b (P-TEFb) for efficient viral gene transcription. Inter- and intra-subtype Tat genetic variation that translates to functional differences has been reported. Specifically, HIV-1 subtype C (HIV-1C) exhibiting Alanine at position 21 of the Tat protein (TatA21) was reported to be associated with reduced LTR transcriptional activity compared to Tat exhibiting Proline at position 21 mutation (TatP21). However, the effect of Tat variation on its ability to recruit P-TEFb is unknown. Therefore, this study seek to determine the effect of HIV-1 subtype C TatA21 mutant on the ability of Tat to recruit P-TEFb to 5’ LTR to enhance viral gene transcription. To this effect, site-directed mutagenesis (SDM) was performed on the Plasmid pcDNA3.1(+) HIV-1C BL43/02 TatA21 to introduce TatP21 alone or together with other mutations using designed primers and the Q5 DNA polymerase kit. The effect of Tat mutations was measured using Tat transactivation assay where the luciferase activity was the measured output in TZM-bl cell lines and the impact of TatA21 was further assessed on ability of the LTR to drive GFP and Gag expression in Jurkat and A72 cells respectively. Next, protein modelling was performed using Hdock software, followed by RNA immunoprecipitation (RNA IP) was performed using stably expressing TatA21 and TatP21 in Jurkat cells. Lastly, co-immunoprecipitation of TatA21 and associated with significantly reduced LTR transcription activity compared to TatP21 (p = 0.0004). TatA21 resulted in had significantly lower GFP expression Jurkat cells (p = 0.0439) and lower Gag expression in A72 cells compared to TatP21. Although TatA21 reduced the LTR transcription activity compared to TatP21, protein modelling using Hdock software revealed that TatA21 and TatP21 protein structures were the same. Consistently, molecular docking showed that TatA21 had a lower binding affinity than TatP21. The RNA IP showed that TatA21 had significantly reduced affinity to bind to TAR compared to TatP21 (p = 0.0151). Moreover, TatA21 and TatP21 formed a complex with cycT1 and CDK9. Taken together, our data shows that HIV-1C TatA21 significantly reduced its transactivation activity but does not affect its ability to recruit P-TEFb. Interestingly, TatP21 is able to bind TAR more efficiently than TatA21 thus revealing a possible mechanism but which the reduced functionality of SDMs and patient derived TatA21 variants was observed. The effect of TatA21 and TatP21 on the propensity of HIV-1 latency development or reversal. To this effect, a recombinant viral vector exhibiting either TatA21 (C731CTatA21C) or TatP21 (C731CTatP21C) were generated. The C731CTatA21C or C731CTatP21C were separately co-transfected together with VSV-G and R8.91 into Jurkat cells for virus production. This virus was then used to infect Jurkat cells for 3 days. Followed by cell sorting of GFP- cells, which represented either truly negative or latently infected cells was then performed. We were able to successfully generate C731CTatA21C virus and characterized it to a 1.2% reactivation. However, the generation of C731CTatP21C recombinant viral vector was unsuccessful and thus could not be used for comparison. Future studies should involve the characterization of TatP21 in the propensity of latency development and/ or reactivation.
Functional characterization of human immunodeficiency virus type 1 (HIV-1) subtype C transmitted/founder (T/F) viruses long terminal repeat (LTR) variants and association with disease outcome.
(2020) Naicker, Shamara.; Madlala, Paradise Zamokuhle.
Background: The persistence of latent viral reservoirs is a major roadblock to human immunodeficiency virus type 1 (HIV-1) cure development. Latent reservoirs harbour transcriptionally silent yet replication competent proviruses. However, the molecular mechanisms that govern HIV-1 latency at the transcriptional level is unknown. Therefore, we hypothesize that HIV-1 subtype C (HIV-1C) transmitted/founder (T/F) 5’ long terminal repeat (LTR) genetic variation may affect disease outcome. Methods: To address this, viral RNA was extracted from plasma samples obtained from 25 HIV-1 infected patients from the HPP and FRESH acute infection cohorts (QIAamp® Viral RNA Mini Kit, Qiagen, Hilden, Germany). Viral RNA was reverse transcribed to DNA using SuperScript™ III One Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, Massachusetts, United States). Nested PCR was performed (Platinum® Taq DNA Polymerase High Fidelity PCR Kit (Invitrogen, Massachusetts, United States) and PCR products cloned into the pGL3 Basic plasmid. LTR/pGL3 recombinant plasmids were sequenced using BigDye Terminator v3.1 Sequencing Kit (Invitrogen, Massachusetts, United States) to confirm correct sequences. The LTR-pGl3 recombinant plasmids were transfected into Jurkat cells alone or co-transfected with either consensus (wild type) subtype C Tat (conTat) or autologous tat (autoTat) to determine the effect of LTR genetic variation on expression of a luciferase reporter gene. Results: Interestingly, our data demonstrate that basal transcription activity significantly differs between LTR variants. Specifically, patients harbouring the Sp1 III: G2A mutation demonstrated significantly lower transcription compared to the wild type LTR. Although conTat co-transfection increased the LTR activity for most of the LTR variants, the T/F virus LTR containing the TATA box mutation (TATAA TAAAA) in combination with other LTR mutations was not induced. Interestingly, the transactivation activity of the autologous Tat was variable among patients. Specifically, the TATA box variant was marginally induced. Lastly, we observed that the majority of LTR variants were more responsive to stimulation by PMA as compared to TNF-α, SAHA and prostratin. Interestingly, our data demonstrate that autologous tat induced transcription positively correlated with viral load at transmission (p=0.0134, r=0.66) and at one-year post infection but was not significant (p=0.3905, r=0.26). Conclusion: These data suggest that the TATAA TAAAA mutation in combination with other LTR mutations may reduce transcription activity. Taken together our data suggest that HIV-1 subtype C T/F viruses LTR genetic variation may modulate viral gene transcription and impact disease outcome.
Genetic and functional diversity of central nervous system (CNS) derived Human Immunodeficiency Virus type 1 (HIV-1) tat from Tuberculous Meningitis (TBM) patients.
(2018) Ramruthan, Jenine.; Madlala, Paradise Zamokuhle.
INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (tat) is a regulatory gene that encodes the transactivator of transcription Tat protein. The Tat effectively increases the activity of the HIV-1 5’ long terminal repeat (5’ LTR) viral promoter to transcribe viral genes. The tat gene has two exons; the first 72 amino acids of Tat are encoded by the first exon, whilst amino acids 73 – 101 are encoded by the second exon. Exon 1 of Tat is sufficient for the transactivation of the 5’ LTR and therefore was the focus of this study. The Tat encoded by exon 1 consists of 5 functional domains these include: the acidic domain (domain I) comprising amino acids 1 – 21, this is a proline rich domain with high sequence variation; the cysteine-rich domain (domain II) comprising amino acids 22–37, is composed of 6 well conserved cysteine residues in subtype C Tat proteins, a mutation at any of the 6 cysteine residue results in loss of Tat activity; the core domain (domain III) comprising amino acids 38–48, is made of a hydrophobic motif and is relatively well conserved. Together, the first 48 amino acids of Tat comprising domains I – III, allow for the transactivation activity of Tat responsible for enhancing viral gene transcription. The basic domain (domain IV) is an RNA-binding domain made up of amino acids 49 – 57 which allows for the binding ability of Tat to the TAR loop structure of the 5’ LTR. Lastly the glutamine-rich domain (domain V) comprised of amino acids 58 – 72, also concentrated with basic amino acids, has the highest sequence variation in Tat. During the early stages of infection, HIV-1 enters the central nervous system (CNS) and replicates at marginal levels compared to high viral replication in the periphery. Yet, there is higher HIV-1 RNA levels in the in the cerebrospinal fluid (CSF) compared to plasma of tuberculosis meningitis (TBM) co-infected patients. However, the mechanisms driving the higher viral replication in the CNS of TBM patients are not well understood. Therefore, the major aim of this study is to characterise genetic and functional diversity of CNS and plasma derived Tat from TBM coinfected patients. We hypothesized that TBM coinfected patients will display genetically distinct HIV-1 tat variants in the CSF as a driver or consequence of higher viral replication in this compartment compared to plasma. METHODS Viral RNA was extracted from matched CSF and plasma samples obtained from 20 HIV- 1 chronically infected patients (17 TBM and 3 non-TBM) using the QIAmp viral RNA Mini kit (Qiagen Inc., Valencia, CA, USA). Extracted viral RNA was reverse transcribed into viral DNA using SuperScript IV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and amplified using two rounds of (nested) PCR with the Platinum ® Taq DNA Polymerase High Fidelity PCR kit (Thermo Fisher Scientific, Boston, MA, USA). Genetic diversity of plasma and CSF derived isolates was assessed in 19 patients (16 TBM and 3 non-TBM) by sequencing, neighbour-joining phylogenetic analysis and both interpatien and intrapatient diversity analysis. The Tat sequences with previously reported mutations that affect Tat function were selected for downstream functional assays. Twelve tat PCR amplicons were cloned into a pTargeT™ expression plasmid (Promega Corporation, Madison, WI). Recombinant pTargeT clones containing patient derived HIV-1 tat was propagated using the QIAfilter Plasmid Maxi Kit (Qiagen Inc., Valencia, CA, USA) to transfect the TZM-bl mammalian cells, which contains the luciferase gene luc under the control of the LTR promoter. A luciferase assay was done to measure the relative luminescence for each Tat mutant and this was correlated to markers of disease progression such as viral load. RESULTS The phylogenetic data from our study show that sequences from plasma and CSF derived HIV-1 tat clustered closely per patient. Genetic variation was seen as varying branch lengths between patient clusters. However, our data do not show significant nucleotide differences between the plasma and CSF tat sequences with a p-distance of 0.059 and 0.062 respectively (p = ns). Additionally, our data revealed that the amino acid sequences were the same between the CSF and plasma compartments, except in 5% of patients that showed differences in positions that were not previously reported to affect Tat activity. However, Tat diversity was observed to occur in all 5 domains of the first 72 amino acids of Tat namely: V4I, P21A, K24S, H29R, S31C, S46Y, R52W, S57R, P59S and D64G. The functional data from our study revealed that most patient derived Tat mutations occurred in combination with other previously reported mutations. Interestingly, Tat mutations that occurred together with P21A in five different patients showed a showed strong positive correlation with CSF viral load in the CNS (p = 0.003; r = 0.98). CONCLUSION We reject our hypothesis that CNS specific Tat mutations were responsible for the high viral load in the CNS of patients who have TBM, as the allele frequencies of reported amino acid substitutions were represented in equal proportions within plasma and CSF derived Tat variants. Furthermore, our functional data shows that majority of all Tat variants from the TBM group had a reduced capacity to transactivate the 5’ LTR. Whilst we cannot confirm that Tat is responsible for the higher viral replication seen in the CNS of TBM coinfected patients, our data demonstrate that all Tat variants with a P21Anmutation significantly correlates to viral replication in the CNS. Future studies should perform site directed mutagenesis to determine the exact mutations that mediate LTR activity.