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Bibliography: p. 189-229.

The human immunodeficiency virus type 1 (HIV-1) uses a mechanism of genetic recoding known as programmed ribosomal frameshifting to translate the proteins encoded by the pol gene. The pol gene overlaps the preceding gag gene in the -1 reading frame relative to gag. It contains neither a start codon nor an internal ribosome entry site (IRES) to initiate translation of its proteins. Rather the host ribosomes are forced to pause due to tension placed on the mRNA when they encounter a specific secondary structural element in the mRNA. This tension is relieved by disruption of the contacts between the mRNA codons and tRNA anticodons at a �slippery� sequence within the ribosomal decoding centre. Re-pairing of the tRNAs occurs in the new -1 frame after movement of the mRNA backwards by one nucleotide, allowing the ribosome to translate the pol gene as a Gag-Pol polyprotein. A change in ratio of Gag to Gag-Pol proteins affects viral assembly, and most significantly dramatically reduces viral infectivity. The prevailing model for the mechanism of -1 frameshifting has focussed on a pre-translocational event, where slippage occurs when the slippery sequence is within the ribosomal A and P sites. This model precludes a contribution from the codon immediately downstream of the slippery sequence leading into the secondary structural element. I have termed this the �intercodon�. Often at frameshifting sites it is a termination codon, whereas in HIV-1 it is a glycine codon, GGG. When the intercodon within the frameshift element was changed from the wild-type GGG to a termination codon UGA, the efficiency of frameshifting decreased 3-4-fold in an in vivo assay in cultured human cells. This result mimicked previous data in the group within bacterial cells and cultured monkey COS-7 cells. Changing the first nucleotide of the intercodon to each of the three other bases altered frameshifting to varying degrees, but not following expected patterns for base stacking effects. Such a result would support a post-translocational model for -1 frameshifting. It suggested that the intercodon might be within the ribosomal A site before frameshifting, and that the slippery sequence was therefore within the P and E sites. This was investigated by modulating the expression of decoding factors for the intercodon - the release factor eRF1 and cognate suppressor tRNAs when it was either of the UGA or UAG termination codons, and tRNA[Gly] for the native GGG glycine codon. These were predicted to affect frameshifting only if slippage were occurring when the ribosomal elongation cycle was in the post-translocational state. Overexpression of tRNA[Gly] gave inconsistent effects on frameshifting in vivo, implying that its concentration may not be limiting within the cell. When eRF1 was overexpressed or depleted by RNAi, significant functional effects of decreased or increased stop codon readthrough respectively were documented. Expression of suppressor tRNAs increased readthrough markedly in a stop codon-specific manner. These altered levels of eRF1 expression were able to modulate the +1 frameshifting efficiency of the human antizyme gene. Overexpression of eRF1 caused significant reduction of frameshifting of the HIV-1 element with the UAG or UGA intercodon. Depletion of the protein by contrast had unexplained global effects on HIV-1 frameshifting. Suppressor tRNAs increased frameshifting efficiency at the UAG or UGA specifically in a cognate manner. These results strongly indicate that a post-translocational mechanism of frameshifting is used to translate the HIV-1 Gag-Pol protein. A new model (�almost� post-translocational) has been proposed with -1 frameshifting occurring for 1 in 10 or 20 ribosomal passages during the end stages of translocation, because of opposing forces generated by translocation and by resistance to unwinding of the secondary structural element. With translocation still incomplete the slippery sequence is partially within the E and P sites, and the intercodon partially within the A site. The nature of the intercodon influences frameshifting efficiency because of how effectively the particular decoding factor is able to bind to the partially translocated intercodon and maintain the normal reading frame.

Bibliography: p. 189-229. / xiii, 230 p., [4] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Public Health, 2002

Thesis (MCur. (Faculty of Health Sciences))--University of Pretoria, 2006. / Summary in English. Includes bibliographical references.

Thesis (Ph. D.)--Georgia State University, 2007. / Title from file title page. Irene Weber, committee chair; John Houghton, Giovanni Gadda, committee members. Electronic text (186 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Dec. 20, 2007. Includes bibliographical references (p. 166-180).

No description available.

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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

Human immunodeficiency virus type 1 (HIV-1) isolates can be differentiated based on their ability to use particular coreceptors – R5 viruses use CCR5, X4 viruses use CXCR4 and R5X4 (dual tropic) viruses use both CCR5 and CXCR4. It is widely reported that HIV-1 subtype C (HIV-1C) has a unique viral coreceptor evolution pattern in that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for this subtype compared to other subtypes. However, dual tropic HIV-1C isolates have occasionally been described. Furthermore, it has been reported that certain highly active antiretroviral drugs (HAART) may select for X4 viral variants. Therefore, this thesis study was undertaken to better understand the functional and genotypic characteristics of dual tropic HIV-1C isolates, and to characterize drug resistance and coreceptor usage patterns in HAART-naïve versus HAART-failing HIV-1C infected patients. Thirty-five functional HIV-1 env clones derived from seven dual tropic HIV-1C strains were generated and their coreceptor usage characterized in transformed cell lines. All 35 env clones efficiently infected transformed cells expressing CXCR4. Twenty of 35 clones (57%) also utilized the CCR5 receptor. No R5-only clones were detected. Functional coreceptor usage data was correlated to env gene sequence data. The ability of the HIV-1C env clones to facilitate infection of primary lymphocytes and monocyte-derived macrophages was next investigated. The majority of clones characterized as X4 or R5X4 on cell lines used either CXCR4 alone or CXCR4 and CCR5, respectively, in primary cells. A few viruses displayed comparable CCR5 and CXCR4 usage and clones from one virus preferred CCR5 usage in macrophages. Thus in a few cases coreceptor phenotyping in transformed cell lines does not predict usage in primary cells. Genetic determinants for coreceptor usage in primary cells require further investigation. Finally the patterns of drug resistance mutations were studied and coreceptor usage among 45 HAART-naïve and 45 HAART-failing HIV-1C infected patients analyzed. Ninety-five percent of HAART-failing patients had viruses with at least one drug resistance mutation. Thymidine analog resistance mutations (TAMs) were present in 55% of patients. HAART-failing patients had significantly higher prevalence (59%) of X4/R5X4-utilizing viruses compared to HAART-naïve patients (30%) (p<0.02) using the Trofile Co-receptor Tropism Assay while 41% of HAART-failing patients used CCR5 and 70% of HAART-naïve patients used CCR5. Functional results correlated with predictive algorithm methods. This study enhances our understanding of HIV-1 pathogenesis and the results have important implications for the use of coreceptor antagonists for the clinical management of HIV-1C infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.