Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.
Role of the leader sequence of human immunodeficiency virus type 1 in viral replication, genome dimerization, encapsidation, and proviral DNA synthesisShen, Ni, 1969- January 2002 (has links)
Human immunodeficiency virus type 1 (HIV-1) genome consists of two identical RNAs that appear noncovalently linked near their 5' ends. The 5' untranslated region is called leader region. The 3' part of leader, i.e. nucleotides U200 to G335 in HIV-1 genomic RNA, between the primer binding site and the gag gene, can fold into 3 stem-loops: the kissing-loop domain (KLD) or stem-loop 1 (SL1), the 5' splicing junction hairpin (SD) or SL2 and SL3. The KLD, from nucleotide (nt) 243 to 277, forms a stem-loop (kissing loop hairpin) seated on top of a small stem bulge (stem B and loop B). The kissing-loop hairpin, or dimerization initiation site (DIS) hairpin consists of stem C and loop C. Loop C contains the autocomplementary sequence (ACS) GCGCGC262 or GUGCAC262, also called DIS. / In the kissing-loop model of HIV-1 genome dimerization, HIV-1 RNA dimerization is initiated by base pairing between the ACS of one RNA monomer and that of an adjacent monomer. / To understand the role of the ACS in HIV-1 replication and HIV-1 genomic RNA dimerization, we replaced the central CGCG261 (or tetramer) of the HIV-1 Lai ACS by other tetramers. Genomic RNAs containing the UUAA tetramer (non-HIV-1 tetramer) were half dimeric, but UUAA genome packaging was unaffected. This was the first evidence that genomic RNA dimerization and packaging can be dissociated (Chapter 2). Destroying stem-loop C reduced genomic RNA dimerzation by ~50%, proviral DNA synthesis by ~85%, and reduced viral infectivity by ~3 logarithmic units. Destroying stem-loop B had similar effects on genome dimerization, reverse transcription, and viral infectivity. We also observed that mutations in stem-loop B and in the DIS hairpin were "non additive" (Chapter 2). / The existence of stem-loop C is supported by phylogenetic evidence, while that of stem-loop B is not, namely, its sequence is completely conserved. We investigated the role of stem B and loop B nucleotides in viral replication, and genomic RNA dimerization. The putative CUCG246/CGAG277 duplex was replaced by 9 alternative complementary sequences, 4 likely to base pair in long (~500 nts) RNAs, as assessed by the algorithm mfold. Among the 4 sequences, 3 preserved genome dimerzation, 1 did not significantly inhibit it, and 2 preserved viral replication. We also asked if 9 deletions or nucleotide substitutions within nucleotide 200 to 242 and/or 282 to 335 could influence genome dimerization. Delta200--226 and Delta236--242 genomic RNAs dimerized relatively poorly despite having neutral or positive influences on stem B, loop B and klh folding (Chapter 3). / Mutations within the Matrix, Capsid, p2 and nucleocapsid genes suppress several functional defects caused by KLD destruction. We tested the effect of these suppressor mutations on genome dimerization and infectiousness of viruses bearing moderate to crippling KLD mutations. Our conclusion is that these suppressor mutations can restore genomic RNA dimerization when DIS is weakened, but not when DIS is denatured or the KLD is destroyed (Chapter 4).
Identification and characterization of HIV-1 specific neutralizing antibodies from HIV-1 seropositive patients and autoimmune (HIV-1 seropositive or seronegative) participants.Naidoo, Thenusha 17 January 2012 (has links)
Since the discovery of HIV-1, the production of an effective prophylactic or therapeutic vaccine remains elusive. An effective vaccine must be able to elicit a potent humoral and cellular immune response. Neutralizing antibodies target the envelope glycoproteins on the surface of HIV-1 virions thereby preventing viral entry. Unfortunately, to date only a handful of neutralizing antibodies have been identified that are capable of neutralizing different viral strains within diverse subtypes, and none have been isolated from HIV-1 subtype C infected patients. In this study, we screened four different HIV-1 subtype C infected patient cohorts for the presence of neutralizing antibodies against a panel of 5 subtype C and 1 subtype B pseudovirus/es in a pseudovirion based neutralizing antibody assay. The CT cohort comprised 9 slow progressor plasma samples, the FV cohort consisted of 11 antiretroviral drug naïve HIV-1 subtype C infected plasma samples. Plasma samples from 10 antiretroviral treatment experienced HIV-1 subtype C infected patients failing first line therapy made up the DR cohort and the JM cohort consisted of 10 serum samples from HIV-1 seropositive or seronegative individuals with an autoimmune disorder. A pseudovirion neutralizing antibody assay was successfully established, and all plasma and serum samples were heat inactivated and screened using this assay. Analysis of the percentage neutralization and IC50 data showed no correlation between the presence of neutralizing antibodies and delayed disease progression in the SP cohort. High levels of neutralizing antibodies were observed in the DR cohort, however future studies are required to confirm if the measured neutralization is due to residual antiretroviral drugs in the plasma or neutralizing antibodies. No samples within the FV cohort showed promising neutralizing antibody activity however the JM cohort harboured 3 serum samples (TN5, TN6 and TN8) that exhibited a greater than average breadth of neutralization and are worth investigating further in future studies. Patients TN5, TN6 and TN8 were all HIV-1 positive with an additional autoimmune disease. The availability of stored bone marrow samples for TN5, TN6 and TN8 will allow for the generation of antibody phage display libraries and isolation of monoclonal antibodies, with potentially broadly cross reactive activity.
Fish, Muhammad Qasim
30 January 2012
M.Sc.(Med.) (Molecular Medicine and Haematology), Faculty of Health Sciences, University of the Witwatersrand, 2011 / The Human Immunodeficiency Virus type 1 (HIV-1) integrase is an essential enzyme required for viral replication. Integrase forms part of an ensemble of proteins known as the preintegration complex and functions by a two-step process. Firstly, the cleaving of the 3’ ends of the viral cDNA genome, known as 3’-end processing. The second step is the insertion of these ends into host DNA by esterification, known as strand transfer. There is no human homologue to integrase which makes it an ideal drug target. However, the strand transfer inhibitor raltegravir is currently the only antiretroviral treatment available that inhibits integrase. The aims of this study were two-fold: firstly to characterise a cohort of South African patients so as to determine the viability of introducing raltegravir as a new treatment option, and secondly, to set up high-throughput integrase inhibitor screening assays (testing integrase enzymatic functionality). An HIV-1 subtype C specific RT-PCR and PCR assay was established for integrase genotyping using 51 integrase inhibitor-naïve patient plasma samples and 22 antiretroviral drug-naive primary viral isolates from South Africa. Seventy-one of the 73 samples were classified as HIV-1 subtype C and two samples were unique AC and CG recombinants in integrase. Amino acid sequence analysis revealed there were no primary mutations (Y143R/C/H, Q148H/R/K, and N155H/S) associated with reduced susceptibility to the integrase inhibitor raltegravir. However, one sample had the T97A mutation, three samples had the E157Q and V165I mutations, and the majority of samples contained the polymorphic mutation, V72I. The expected finding of no major integrase mutations conferring resistance to integrase inhibitors suggests that this new antiretroviral drug class will be effective in our region where HIV-1 subtype C predominates. However, the impact of E157Q and other naturally occurring polymorphisms warrants further phenotypic investigation. The integrase sequence of viral isolate, FV3, was closest to the consensus sequence, and thus chosen for preintegration complex isolation for use in strand transfer assays. Isolation of preintegration complexes following FV3 infections of several cell lines was unsuccessful as determined by western blot analysis. Subsequently, the focus was changed to isolation of HIV-1 subtype B recombinant integrase and its functional evaluation. Expression of native integrase (INwt) and soluble integrase (INsol) was induced in E. coli, and both proteins were purified by nickel chelating chromatography. The purified recombinant proteins were used to develop three assays to test for strand transfer activity, of which two were successfully established. Furthermore, only INsol showed strand transfer activity in the high-throughput microtitre plate assay and scintillation proximity assay (SPA)-bead strand transfer assay. Activity of INsol was shown to be inhibited by the control compound, chicoric acid with an IC50 of 101.5nM in the high-throughput microtitre plate assay, whereas INsol activity as well as a dose response to chicoric acid with an IC50 of 248.5nM was recorded in the SPA-bead strand transfer assay. Visualization of radiolabelled enzymatic products of strand transfer by polyacrylamide gel electrophoresis of urea sequencing gels was unsuccessful. Overall, the high-throughput microtitre plate and SPA-bead strand transfer assays have been successfully established in our laboratories, and are available to screen compound libraries for potential antiretroviral drug candidates targeting integrase strand transfer.
Differential timing of translocation of HIV-1 subtype B and C Vpu to the ER/Golgi an plasma membrane compartmentsBell, Catherine Macdonald 19 April 2010 (has links)
MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, 2009 / The HIV-1 Vpu protein functions largely to target CD4 molecules for proteasomal degradation, and to enhance virion release. The subcellular localisation of Vpu is related to these functions. Previous studies showed subtype B Vpu localisation at the ER/Golgi complex, while subtype C Vpu localised to the plasma membrane (PM) at 48 hours post-transfection. To determine if subtype C Vpu can localize to the ER/Golgi, we evaluated the cellular localisation of Vpu from two South African subtype C isolates as compared to subtype B Vpu, over time. Codon optimized vpu genes from subtype C isolates FV5 and FV15 (which have a six and two amino acid insert in the N-terminal domain, respectively) and a representative subtype B vpu were TA cloned into the pcDNA6.2/C-emGFP expression vector. The three VpuemGFP recombinant plasmids were cotransfected with pDsRed-ER, pDsRed-Golgi, or pDsRed-Mem into HEK 293T cells, and observed at 24, 48, and 60 hours posttransfection under a confocal microscope to confirm the presence of Vpu at different subcellular compartments. Cotransfection and microscopy conditions were methodically optimised. At 24 hours post-transfection, the subtype C FV5 Vpu had ER/Golgi localisation, but none at the PM. The subtype C FV15 Vpu had weaker ER/Golgi localisation and no PM localisation. In contrast, the subtype B Vpu had strong PM localisation. At 48 hours, FV5 and FV15 Vpu showed PM localisation, while subtype B Vpu was clearly localised at the ER/Golgi. At 60 hours, FV5 Vpu was observed at the PM, whereas FV15 and subtype B Vpu showed ER/Golgi localisation. These findings illustrate the efficient translocation of Vpu between different cellular compartments and for the first time, the difference in timing between subtype B and C Vpu, as well as íntrasubtype differences. This difference in shuttling suggests implications for the timing of viral assembly and release. Further investigations may clarify the impact of this timing on the difference in disease pathogenesis noted between infections with the different subtypes.
Η έκφραση του ογκογονιδίου ets-2 σε υποπληθυσμούς Τ λεμφοκυττάρων και ο ρόλος του στην μεταγραφή του γονιδίου της ιντερλευκίνης-2 και του ιού HIV-1Παναγούλιας, Ιωάννης 25 July 2008 (has links)
Η έκφραση του ογκογονιδίου ets-2 δρα κατασταλτικά στην έκφραση του γονιδίου της ιντερλευκίνης-2 καθώς και στην έκφραση του ιού HIV-1 στα παρθενικά Τ λεμφοκύτταρα. / -
Structural and functional investigation of human chemokines and applications of human chemokines in blocking HIV-1 entryJin, Hongjun 15 May 2009 (has links)
Chemokines are important mediators of leukocyte migration. Chemokines bind to G protein–coupled receptors (GPCR) and cause conformational changes that trigger intracellular signaling pathways involved in inflammation, injury healing, cancer, metastasis, and HIV infections. No direct structural information about any chemokine receptor is available, but the structure of chemokines has been well studied. Structural studies of chemokines coupled with cell-biological investigations may lead to a better understanding of the mechanisms of chemokine-receptor interactions. In this Ph.D. project, I studied the structural and functional relationship between chemokines and chemokine receptors using NMR, X-ray crystallography, and mutagenesis approaches, coupled with several different cell-biology assays. We found that the conserved “chemokine fold” can support different dimerization types in the chemokines family, although changing the dimers from CC- to CXC-type fold is not readily accomplished. I also used an engineered covalently-bound dimer of the MIP-1β mutant, MIP-1β-A10C, to study the relationship between dimerization of chemokines and their interaction with the CCR5 receptor. My results suggest that MIP-1β dimer neither bind nor activate the CCR5 receptor. I also studied the biophysical properties of one N-terminal awkward mutant of P2-RANTES, which was originally selected by others from a phage display using CCR5-expressing cells. Although the NMR and X-ray crystal studies revealed that the wild type RANTES is a tight homodimer, analytical ultracentrifugation reveals that P2-RANTES is a monomer in solution, the 1.7 Å resolution X-ray crystal structure of P2-RANTES was found to be a packed tetramer. The mutated N-terminal residues play a very important role in the tetramerization in the X-ray crystal structure. Finally I used the HIV-1 env mediated cell-cell fusion assay to study the combination of chemokines or chemokine variants with anti-HIV peptides C37 or/and T-20. A surprisingly synergistic effect was found between P2-RANTES and C37 or T-20. This combination stratagem may lead to further useful drug combinations or drug delivery for more potent anti-HIV treatments.
Parvez, Md. Kamal Uddin
12 April 2011
HIV-1 integrase (IN) enzyme employs several viral and cellular proteins for nuclear translocation and crucial integration of viral cDNA. Successful identification of new viral/cellular interactions may shed light for better understanding of HIV-1 replication. 293T cells were transiently transfected with pYEF-1-TAP-IN and cell lysate were subjected to Tandem Affinity Purification system to pull down putative IN-interacting cellular partners. A number of distinct bands from the Coomassie-stained gel were excised followed by in-gel digestion and mass spectrometry. Putative cellular partners of HIV-1 IN were heat shock protein 60 (HSP60), β-tubulin, γ-actin, ATP synthase alpha subunit and histone H1.2 were identified by mass spectrometry. Additionally, SF3A3 (splicing factor 3A3), another previously reported factor, was successfully co-immunoprecipitated with IN. The C-terminal portion of IN was found to be the region of interaction with SF3A3. Overall, this study has provided better understanding of IN dynamics enriching existing knowledge of HIV-1 IN biology.
Proviral HIV-1 hypermutation: the correlation of APOBEC3G/F and HIV-1 Vif in HIV-1 disease progressionDe, Sujata Monika 12 April 2011 (has links)
APOBEC3 proteins, in particular APOBEC3G/F, are important innate host factors that contribute to protection from HIV-1 infection by inducing high levels of guanine to adenine nucleotide substitutions (termed hypermutation) during HIV-1 viral replication. These nucleotide substitutions occur at different rates and locations across the HIV-1 genome and are thought to be particularly more frequent in the pol region. The virus has evolved ways to counteract these host factors by inducing degradation of APOBEC3G/F proteins through protein interactions with HIV-1 Vif. The aim of this thesis is to characterize and investigate the role of APOBEC3G/F-mediated hypermutation in the HIV-1 genome. We identified a subset of women from the Pumwani Commercial Sex Worker (CSW) cohort with significantly higher rates of hypermutated proviruses in pol. This degree of hypermutation correlated to less severe HIV disease progression as measured by CD4+ T cell count. This was in agreement with previous studies that evidence of APOBEC-mediated hypermutation correlate with reduced disease progression, confirming APOBEC3G/F proteins role in HIV-1 disease. Furthermore, we investigated the in vitro and ex vivo interaction between HIV-1 Vif and APOBEC3G from subjects infected with hypermutated and non-hypermutated proviruses. In vitro studies indicated that HIV-1 Vif protein expression in subjects with hypermutated proviruses were quite divergent and levels of APOBEC3G also differed between subjects. Ex vivo studies in subjects with hypermutated proviruses indicated that endogenous APOBEC3G expression was greater than in subjects with hypermutated proviruses. Both studies suggest that host and viral factors such as APOBEC3G and HIV-1 Vif are playing an influential role in HIV-1 pathogenesis. Further investigations into these interactions may lead to novel strategies into the development of therapeutic drugs for the fight against HIV-1.
Measurement and characterization of HIV inhibitory Clade A Serpins in the cervical mucosa of highly HIV-1 exposed seronegative individualsRahman, Syeda Sharmin 04 November 2011 (has links)
Objective: Serpins are serine protease inhibitors that are involved in a wide variety of biological functions in nature. They are known to regulate inflammation processes as well as provide host defense against microorganisms. Recent evidence has associated many types of mucosal serpins with a protective phenotype against HIV infection in women. Our hypothesis is that serpins with known antiviral activity against HIV-1 are correlated with protection in a group of HIV exposed seronegative individuals (HIV-resistant) from the Pumwani sex worker cohort. Study design: Cervico-vaginal lavage (CVL) fluid was collected from 66 HIV-positive, 82 HIV-negative and 84 HIV-resistant sex workers from the cohort. Clinical and epidemiological information was recorded at the time of sample collection. CVL protein levels were determined by BCA assay and serpin (A1 and A3) concentrations by a commercially available ELISA kit. Mucosal serpin concentrations were compared against clinical and epidemiological factors as well as sexual practices. Results: Serpin A1 was significantly higher in the HIV-resistant group compared to the HIV-negative controls (Anova: p=0.0470*). Total concentration of serpin A3 did not reach statistical significance between groups. Serpins did not correlate with age, sexual practices, contraceptive use or number of pregnancies. Serpins were differentially abundant during different stages of the menstrual cycle whereas serpin A1 was elevated during the proliferation phase but not in secretory phase (p=0.0275*). Conclusion: Serpin A1 was correlated with HIV-protection in this group of HESN women. This work will contribute to a more complete understanding of mechanisms of resistance and susceptibility to HIV infection.
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