Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / The HIV-­‐1 pandemic continues unabated. Although treatments exist that can substantially alleviate the morbidity and mortality associated with HIV, there is still a need for improved anti-­‐HIV treatments that reduce toxicities and administration frequency and mediate sustained inhibition of viral replication. Given the high mutability and variability of the virus, a strategy that is garnering increasing focus is the targeting of host factors that the virus requires to replicate, so-­‐called HIV-­‐dependency factors (HDFs). It is hoped this will reduce the emergence of viral drug resistance. A number of genome-­‐wide screens have been performed to identify HDFs, although many remain to be validated, particularly in relevant cells lines. An objective of this thesis was to validate three host factors as HDFs, in both TZM-­‐bl reporter and T cell-­‐derived cell lines, and to examine their potential as anti-­‐HIV-­‐1 therapeutic targets through exploitation of the cellular gene silencing pathway, RNA interference (RNAi). These were HIV-­‐1 Tat specific factor 1 (HTATSF1), DEAD (Asp-­‐Glu-­‐Ala-­‐Asp) box polypeptide 3, X-­‐ linked (DDX3X) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1 (SMARCB1), selected because they had been previously implicated in HIV-­‐ 1 pathogenesis. The well-­‐characterised HDF, PC4 and SFRS1 interacting protein 1 (PSIP1)/lens epithelium-­‐derived growth factor (LEDGF)/p75, was included in the study as a positive control. Cassettes expressing short hairpin RNAs (shRNAs) targeting the four host proteins were generated, although shRNAs did not suppress endogenous ddx3x mRNA levels. The ability of shRNAs to inhibit HIV-­‐1 replication in the reporter cell line, TZM-­‐bl, was examined. These HeLa-­‐ derived cells are permissive for R5-­‐tropic HIV-­‐1 infection and contain an integrated luciferase gene driven by the viral promoter. shRNAs mediated a dose-­‐dependent inhibition of luciferase activity in cells infected with a HIV-­‐1 subtype B molecular clone and, although production of the viral protein p24 was unaltered, infectious particle production was decreased in cells treated with a shRNA suppressing HTATSF1. Little effect was observed with a shRNA targeting SMARCB1, suggesting that this may not function as an HDF under these conditions. No effect on infectious particle production was seen with the shRNA targeting PSIP1, which was a result of the long half-­‐ life of this protein, highlighting a limitation of using such reporter systems for HDF validation. Importantly, shRNAs were not associated with any cytotoxic effects in TZM-­‐bl cells. Whether HTATSF1 is a potential therapeutic target was interrogated further in the more relevant T cell-­‐derived SupT1 cell line. Lentiviruses were used to generate populations where >90% had one copy of the integrated shRNA expression cassette. Replication of the subtype B molecular clone p81A-­‐4 was significantly inhibited in the shH1-­‐expressing SupT1 cell line, which targets HTATSF1, for over 14 days post-­‐infection, although inhibition was not as pronounced asthat observed in the shP1-­‐expressing SupT1 cell line, which targets PSIP1. In contrast to a previous report, no change in the ratio of unspliced to singly-­‐ or multiply-­‐spliced HIV-­‐1 transcripts were detected in shH1-­‐expressing SupT1 cells, suggesting that HTATSF1 does not function as a splicing cofactor in this system. A slight rebound in p24 levels at 14 days post-­‐infection was accompanied by increased HTATSF1 expression and a decrease in the percentage of cells with transgene expression in the population. In addition, there was a slight decrease in shH1-­‐derived guide strand expression, but no change in transcription rates of the htatsf1 gene, suggesting that cells within the population with shH1 expression and HTATSF1 suppression may have a growth disadvantage. Thus, although this work demonstrates for the first time that HTATSF1 functions as an HDF in T cell-­‐derived SupT1 cells, it may not constitute a viable therapeutic target. A second objective of this thesis was to examine the feasibility of transcriptional gene silencing (TGS) of HDFs as an anti-­‐HIV strategy. TGS is a small RNA-­‐induced gene silencing pathway that operates through chromatin remodelling with the potential to mediate long-­‐term silencing of gene expression. Thus, its application may reduce the frequency of drug administration and associated toxicities. Short interfering RNAs (siRNAs) targeting the htatsf1 promoter were able to reduce target mRNA expression, which was accompanied by decreased htatsf1 transcription rates in HEK293T cells, suggesting silencing via a TGS mechanism. The htatsf1 silencing inhibited infectious HIV-­‐1 particle production from TZM-­‐bl cells. This work provides proof of principle that TGS induction at a HDF may inhibit HIV-­‐1 replication. siRNAs targeting the ddx3x promoter did not induce TGS. To examine whether gene susceptibility to TGS may be influenced by promoter architectures, 49 promoter features were examined for enrichment in genes at which small RNA-­‐induced TGS has been reported. Initially, the TGS group was compared to a random set of 2,000 promoters and then all other promoters in the genome. To control for gene activation, two further analyses were performed comparing the TGS group features to those from promoters active in the THP-­‐1 cell line and housekeeping genes. Whilst difficult to ascribe differences between the TGS group and the control groups to anything beyond a variation in the proportion of active genes within each group, there was enrichment for certain promoter features that are independent of activity; the TGS group was characterised by broad transcription start regions, high CpG content and a single expression profile. Moreover, the fraction of promoters with reported non-­‐coding RNA overlap was greater in the TGS group than the control groups. Thus, there is some evidence that a number of promoter features are associated with TGS susceptibility. It is hoped this novel analysis will facilitate selection of future TGS targets, including HDFs. In summary, the work presented in this thesis paves the way for development of improved anti-­‐HIV therapies involving HDF-­‐targeted TGS-­‐based gene therapies that mediate sustained inhibition of the virus.

Reverse transcription in HIV-1 (human immunodeficiency virus type 1) is initiated from a tRNA, tRNALys3, that is annealed to the primer binding site (PBS) in the 5' region of viral RNA. This tRNA, along with the other major tRNALys isoacceptors, tRNALys1,2 , is selectively packaged into HIV-1 during its assembly. The formation of a tRNALys packaging/annealing complex is believed to involve the interaction between a Gag/GagPol/viral complex with a lysyl-tRNA synthetase (LysRS)/tRNALys complex, with Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys. In fact, Gag particles alone will package LysRS, but GagPol, which binds tRNA Lys, is also required for incorporation of the tRNALys. / The model we propose for the tRNALys packaging/annealing complex predicts a possible interaction between LysRS and Pol sequences in GagPol, which might facilitate transfer of tRNALys3 from LysRS to the reverse transcriptase (RT) thumb domain where tRNALys3 binds. In this work, we demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. Since these RT domains are not required for tRNALys packaging into HIV-1, the LysRS/Pol interaction is probably not involved in the transfer of tRNALys3 to RT. The LysRS/Pol interaction may instead be involved in tRNALys3 annealing since the connection domain in RT has been found to be required for this process. Also, since an interaction has been reported between Gag and Pol sequences in GagPol, we also investigated whether the Gag/LysRS/Pol interaction played an important role in stabilizing the Gag/Pol interaction, and found, using siRNA to LysRS, that it did not. / tRNALys3 annealing to viral RNA is promoted by nucleocapsid sequences in Gag and by mature NCp7, and we have examined the roles of Gag and NCp7 in this process. Gag- and NC-facilitated tRNALys3 annealing to HIV-1 RNA were measured both in vivo and in vitro, indirectly by the ability of annealed tRNALys3 to prime reverse transcription, and directly by measuring the occupancy of the PBS by tRNALys3. While tRNALys3 annealing can be carried out by both Gag and NCp7, exposure (in vivo or in vitro) of the tRNALys3/viral RNA complex to NCp7 is required for optimum placement of the tRNALys3. This is indicated by 1) tRNALys3's reduced ability to incorporate the first dNTP, dCTP, and 2) its more ready displacement from the PBS by DNA synthesized from a downstream primer. / It has been previously demonstrated that APOBEC3G (A3G) can inhibit tRNA Lys3 annealing to viral RNA, and we have used A3G to further dissect the roles of Gag and NCp7 in annealing, both in vitro and in vivo. Experiments studying how APOBEC3G (A3G) inhibits tRNA Lys3 annealing indicate that in protease-positive viruses, Gag-facilitated tRNALys3 annealing may only playa minor role. In vivo and in vitro, A3G only inhibits NCp7-facilitated annealing, and not Gag-facilitated annealing. Nevertheless, while Gag is able to show 70-80% of the annealing efficiency of NCp7 in a protease-negative virus, A3G can reduce annealing efficiency in protease-positive viruses to 40%. This appears to be due to the fact that, in vitro, the presence of NCp7 makes prior Gag-facilitated annealing susceptible to A3G. This suggests that in wild type viruses, any Gag-facilitated annealing of tRNALys3 to viral RNA that does occur is altered through an A3G-susceptible re-annealing by NCp7.

The overall aim of this project was to investigate HIV-1 genetic diversity in South Afri ca and to characterise the immune response in mice to a South African subtype C gp120. To investigate the relationship between subtype and mode of transmission, samples were collected from individuals infected by heterosexual and male homosexual transmission from patients attending local HIV/AIDS clinics in Cape Town (n=49) and Bloemfontein (n=4). Isolates were subtyped using heteroduplex mobility assay (HMA) based on the V3-V5 region of the env geneusing reference plasmids (2 B, 2 C and 1 D) representative of local subtypes. HMA identified four env subtypes: A, B, C and D. Subtype B viruses were found in 92.9% (26/28) of the male homosexual/bisexual group and subtype C viruses in 77.2% (17 /22) of the heterosexual group. Subtype B viruses were also identified in two heterosexual patients, one patient infected by blood transfusion and in two patients with. unknown mode of transmission. Subtype D viruses were found in one male homosexual patient and one heterosexual patient and a husband and wife couple were infected with subtype A viruses. A significant association between subtype and mode of transmission (p=<0.0001) was identified, confirming two independent epidemics. To determine the subtype distribution of HIV within urban heterosexual populations throughout South Africa, samples were collected from women attending antenatal clinics in Johannesburg (n=34), Pretoria (n=S) and Durban (n=20). Samples from Bloemfontein (n=24) were taken from individuals attending an HIV/AIDS clinic. All eighty-three samples were subtyped by HMA in the env region as before. The predominant subtype circulating within the urban heterosexual population throughout South Africa was identified as subtype C (92.8%) although subtype B was also detected (7.2%). It may thus be beneficial if a HIV vaccine for South Africa is based on a subtype C model. In addition, a rapid method for identification of HIV-1 gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400bp (p17) or 650bp (p17 and 5' p24) long PCR fragments. This strategy was appl i ed to eighty-six samples (Cape Town n=47, Johannesburg n=20, Bloemfontein n=17 and Durban n=2) previously subtyped by either sequence analysis of the gag p17 region (n=31), heteroduplex mobility assay (HMA) based on the env gene (n=76), or both (n=21). RFLP analysis identified two subtype A, twenty-five subtype B, fifty-eight subtype C and one subtype D isolates. There were no discrepancies between RFLP and sequence gag subtypes, demonstrating the reliability of this method and no discordance between gag RFLP subtypes and env HMA subtypes, indicating no recombinant viruses in the genomic regions analysed.

No description available.

Human Immunodeficiency Virus type I genome consists of two identical RNA molecules that are non-covalently linked to form a dimer. HIV-1 immature and mature genomic RNA (gRNA) dimers were found in protease defective (PR -) and wild type virions, respectively, and the 5'untranslated region (5' UTR) was shown to play key roles during the genome dimerization process; but the dimerization mechanism still remains to be clarified My research project is to characterize the dimerization process and the role of 5' UTR in genome dimerization in virions produced by tissue culture cells. I'll firstly show the dimer maturation processes of HIV-1 gRNA isolated from newly released to grown-up (≥10h old) wild type, PR-, and SL1 defective (DeltaIDS) virions respectively. The results showed that HIV-1 gRNA dimer maturation process was protease-dependent and involved multiple steps: from low to high dimerization level and dimer thermostability, and from low dimer mobility to intermediate and high mobility. PR- virions did not freeze gRNA conformation in the primordial nascent state and gRNA changed from monomeric in newly released virions to half dimeric in grown-up virions, which showed that genome was packaged in the form of monomeric RNA or fragile dimers, more thermolabile than immature dimers in grown-up PR- virions. DeltaDIS inhibited gRNA dimerization by about 50% in newly released virions, though grown-up DeltaDIS gRNA was fully dimeric, which indicated that the DIS played the initiation role in gRNA dimerization in HIV-1 virions. The gRNA dimerization rate in PR- or DeltaDIS virions was much slower than that in wild type virions. These results show for the first time the whole process of dimer maturation after virion release, the gRNA conformation rearrangement in PR- virions, and the initiation role of the DIS in HIV-1 virions. Next, I'll provide a rather systematic search for the contribution of different regions in 5' UTR to HIV-1 gRNA dimerization by studying selected mutations singly or together with defective SL1. The results showed that the 5'trans-activation response element (5'TAR) was directly involved in gRNA dimerization, and a long distance base-pairing interaction between a sequence in U5 region (nts105-1l5) and another around the initiation codon of the gag gene (nts334-344) was structurally contributive to gRNA dimerization. Deletions of sequences around the 3'end of Primer Binding Site (PBS) stem-loop moderately decreased gRNA dimerization level. Other sequences in 5' UTR except DIS/SL1, which was previously known to play important roles, didn't show any systematic role. Here the results suggested that the absence of inhibition on gRNA dimerization level with defective DIS might be the compensation of the direct role of 5'TAR; and wild type-like dimerization level of DeltaTAR must be the direct contribution of the DIS.

Paediatric human immunodeficiency virus (HIV) infection has become a major socio-economic health problem in recent years as the number of HIV-1 infected children steadily increases. The majority of these infants are infected through mother-to-child transmission, with the frequency of vertical transmission varying between 12,9% and 65%. In order to implement appropriate management and possible treatment of these infected neonates, it is essential to have reliable laboratory tests for the early diagnosis of an HIV infection. At the time that this study was initiated, the diagnosis of HIV-1 infection in the Groote Schuur Hospital Virology Laboratory depended almost exclusively on serological assays. Such assays are of limited value for infants under 18 months of age, as maternal lgG antibody to HIV-1 is transferred via the placenta and may persist in the baby for up to 18 months. Available lgG antibody tests do not distinguish reliably between passively acquired maternal antibody and that produced by the infant itself. A valuable method of establishing the presence of true infection is provided by the polymerase chain reaction (PCR) technique which allows the identification, and subsequent exponential amplification of low levels of specific viral nucleic acid using specific oligonucleotide primers. A major aim of this study was to develop and instigate a (PCR) assay for the early diagnosis of HIV infection in infected infants. This was successfully achieved by the adaptation and optimization of an existing standard PCR protocol to suit the specific needs of a routine diagnostic service. Preliminary requirements involved the selection of primers and probes and establishing optimal parameters for: ionic strength, Taq DNA polymerase concentration, primer concentration, deoxynucleotide triphosphate concentration, and hybridization conditions for most efficient functioning of the test. The devised method entailed the extraction of proviral DNA from peripheral blood mononuclear cells, amplification of HIV-1 specific sequences by PCR, and identification by Southern blot hybridization with digoxigenin (DIG)-labelled probes. Thereafter the efficacy of the assay was tested on 45 infants (under 15 months of age) all born to seropositive mothers and therefore at risk for HIV infection. Forty-two of these infants had antibodies to HIV-1 and the remaining 3 were seronegative. The latter 3 also tested negative for HIV proviral DNA when PCR was performed, using at least 2 different HIV-1 primer pairs and their respective DIG-labelled probes. However, 27 (64%) of the 42 seropositive infants were also HIV-PCR positive and the remaining 15 (36%) seropositive infants were negative for HIV proviral DNA. Positive PCR tests correlated well with clinical data indicative of active HIV-1 infection for the majority of infants in the neonatal period, although it could not provide proof of infection in newborn babies (less than 1 week of age). The development of an in-house PCR protocol specific for HIV-1 has not only provided a valuable diagnostic assay for neonatal infection, but has also given insight into the parameters required for high sensitivity and the stringent precautionary measures that need to be applied to avoid contamination problems. The second part of this study was devoted to DNA sequence analysis of cloned HIV isolates from an infected mother and her 3-month-old infant. Nucleotide sequence variation between isolates of HIV-1 has been well documented. Examination of the third variable region (particularly the V3- loop) in the env gene of HIV-1 of our mother-infant pair confirmed this variation and provided the first genetic epidemiological data of this nature in the local community. Proviral DNA from both mother and baby was amplified using V3-specific degenerate primers and cloned. Clones containing the insert DNA were 2 identified by colony-blot hybridization. Their nucleotide and amino acid sequences were analyzed by using various computer programs. The degree of similarity between variants from the mother and infant in this study differed to a large extent from previous studies. The virus population harboured by the mother displayed highly homogeneous V3 sequences (1,04% variation) compared to the isolates from her 3-month-old infant, which showed a higher degree (1,8%) of heterogeneity. Phylogenetic analysis of the different isolates from mother and infant demonstrated that an HIV-1 subtype C virus was the infectious agent. This classification was confirmed by the characteristic amino-acid sequence of the tetrapeptide motif of the V3 loop present in the isolates from both mother and infant as well as the absence of a potential N-linked glycosylation site proximal to the first cysteine of the V3 loop, which is characteristic of subtype C viruses. Based on the amino acids present at positions 306 and 320 of the V3 loop, it could also be concluded that isolates from both the mother and her baby were consistent with the non-syncytium inducing (NSI) phenotype of HIV-1, thus indicating that, contrary to popular belief, NSI variants can be responsible for initiating infection. Data obtained from these genetic investigations of variants involved in vertical transmission of HIV-1 can form a useful basis for future comparative studies.

No description available.