Sequence analysis of human immunodeficiency virus type 1: A cross-sectional and longitudinal analysis

The replication of HIV-1 and the turnover of CD4 cells in vivo is a highly dynamic process. This environment of continuous de novo virus infection and extensive replication provides an ideal setting for the generation of HIV-1 variants. The objectives of this thesis were to perform a cross-sectional and longitudinal sequence analysis of HIV-1 in blood and multiple tissues of HIV-1 infected individuals. Five to 20 clones of Polymerase Chain Reaction (PCR) amplicons were sequenced and their relationships investigated using phylogenetic methods. An analysis of V3 sequences from a patient at the time of seroconversion revealed a homogeneous quasispecies which contrasted with the high diversity observed in the V3 quasispecies from a patient with AIDS. Phylogenetic analysis of HIV-LTR variants from LN and blood from 4 patients revealed 2 distinct patterns. First, an independent clustering of LN and PBMC variants, indicating a compartmentalisation of the two quasispecies for the patient with an intact lymph node. Second, non-polarised trees with variants from either LN or PBMC present in common branches for the patients with a disrupted LN architecture. These results suggest that the lymph nodes may be important in the sequestration/evolution of distinct HIV-1 variants. A similar phylogenetic analysis has been carried out on LTR variants from multiple postmortem samples (spleen, lung, spinal cord, ganglion, lymph node and blood) of a patient who died with AIDS. The results revealed a genetically distinct LTR quasispecies in the nervous tissues compared to that present in the other tissues. The nervous tissue quasispecies was characterised by 7 mutations in the normally conserved TAR region which would be expected to abrogate tat transactivation. However, this quasispecies showed the maintenance of prototypic NF-kB sites which would allow TAR independent kB tat transactivation to occur. These data provide evidence for a role of the LTR in the adaptation of HIV-1 to the nervous tissue environment by optimising the use of specific cellular transcription factor(s)