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Infection and replication
The membrane bound protein, CD4, acts as a primary ligand for the HIV glycoprotein gp120[Kwong et al., 1998]. Attachment of gp120 is followed by attachment of the fusion protein, gp41 to either CCR5 or CCRX4. This binding induces fusion of the viral envelope with the target cell membrane, and is followed by internalisation of the viral particle [Wyatt and Sodroski, 1998]. After entry, the viral core particle and nucleocapsid undergo proteolysis, releasing the RNA copies of the HIV genome as well as accessory proteins, i.e. integrase, protease and reverse transcriptase [Auewarakul et al., 2005]. The RNA genome is reverse-transcribed by RT and the product can be inserted into the host cell’s genome by integrase [Chiu and Davies, 2004]. This integration into the host genome allows perpetuation of the virus along with the T-cell upon cell replication. The expression of the whole HIV genome usually occurs in conjunction with the acti vation of the infected cell, notably under the influence of the transcription factor NF-κB, which is expressed at high levels after T-cell activation [Duh et al., 1989]. Initially, the mRNA transcript that consists of the entire HIV RNA genome is spliced, producing tran scripts coding for HIV proteins [Felber et al., 1989]. This includes Rev, which allows the transport of the entire HIV RNA transcript to the cytosol intact [Fischer et al., 1995]. The gag and env protein products are produced.
Loading of peptides onto major mistocompatibility class I
The MHC Class I molecule is the final step before the peptide is presented on the cell membrane. The binding site for the peptide consists of a bed of anti-parallel β-strands, overlaid and flanked by two α-helices [Li and Raghavan, 2010]. The peptide ligand binds in the groove formed by the alpha helices, as shown in Figure 1.10 on the preceding page. There are six MHC class I genes, termed Human Leukocyte Antigen (HLA) in humans, HLAs A, B, C, D, E and F. Of importance here, are the classes A, B and C, which bind peptides from intracellular proteins. HLA molecules are extremely polymorphic and thousands of HLA allotypes are known, the most polymorphic being HLA A, B and to a certain extent, HLA C [Jin and Wang, 2003]. The polymorphisms determine the binding motif for each HLA type and thus the composition of the peptide determines the affinity of it to MHC. The MHC binding groove contains position-specific binding pockets that have strong affinities for a specific range of residues. It preferentially binds peptides that are nine amino acids in length, but peptides with lengths ranging from 8-11 are not uncommon. During motif discovery experiments, it was revealed that there exist 2-3 pockets that can bind a limited range of amino acids [Rammensee et al., 1995]. These positions are called anchor positions and it is essential that the correct amino acids exist in the correct position in order for the potential ligand to bind strongly to the groove. The binding groove of the HLA A*0201 molecule with bound peptide LLFGYPVYV is shown in Figure 1.10 on the previous page. From the clefts on either side of the binding groove, it is easy to see that the length of the binding peptide is limited. The peptide needs to span a majority of the groove to form interactions with crucial binding pockets which exist near the clefts. It has been demonstrated that when the P1 residue of the peptide is removed, binding of this peptide to the groove still occurred, but at a severely lowered affinity [Khan et al., 2000]. Empty MHC Class I molecules do not form stable complexes and are not presented on the cell membrane, ensuring that there is no wastage. Binding of an MHC ligand changes the conformation of the groove from an open to a closed state. This allows the MHC-peptide complex to have a long half-life, sometimes even tens of hours [Khan et al., 2000]. The long exposure grants a higher probability that a circulating CTL with a complementary TCR encounters the peptide. Figure 1.11 on the following page illustrates binding motifs for HLA allotypes A*0201 and B*5801. The figure depicts positions of interest along a typical nonamer peptide.
1 Introduction
1.1 Human Immunodeficiency Virus
1.2 The adaptive immune response and the influence of HIV infection
1.3 Antiretroviral therapies
1.4 Vaccines for HIV
1.5 Pharmacological and immunological interaction
1.6 Summary
2 Problem statement and Aims
2.1 Problem statement
3 Methods
3.1 Acquisition and processing of HIV sequence data
3.2 Imputation of HLA types
3.3 Using Fisher’s exact test to calculate substitution discrepancies
3.4 Calculating epistatic interactions between substitutions within PR and RT
3.5 Prediction of peptide CTL epitope eligibility
3.6 Accounting for Epistatic Interactions to Compensate for Bias
3.7 Reconstruction of ancestral states
3.8 Summary
4 Results
4.1 Interaction between HLA type on antiretroviral resistance mutation frequencies in HIV subtype B
4.2 Exploring causal relationships between HLA types and diminished ARV resistance-related substitutions
4.3 Evidence of multiple diminished antiretroviral related pol substitutions in HLA type B*15 and B*48 assigned sequence sets
4.4 Binding motifs may reveal a mechanism for similar CTL epitope presentation of HLA types B*15 and B*48
4.5 The viability of the peptides LM9 and RQ10 as epitopes
4.6 Evidence of higher selection pressure in the predicted epitope regions
4.7 Concerning HIV subtype C
4.8 Summary
5 Conclusionary Discussion
5.1 Fitness and genetic hurdles of ARV resistance acquisition under induction of CTL response
5.2 The importance of the substitutions PR 90M and RT 215Y/F
5.3 Interaction between HLA and antiretroviral resistance mutations in light of vaccine design
5.4 Further improvements of CTL epitope detection in HIV strains with acquired antiretroviral resistance
5.5 Conclusion
