The role of cellular lipids in HIV-1 replication.

The role of cellular lipids in human immunodeficiency virus type-1 (HIV-1) replication is well established. There is also a large body of evidence that indicates that HIV-1 utilizes lipid rafts at the plasma membrane for viral entry, assembly and budding. HIV-1 is enriched in cholesterol and sphingomyelin which are two of the major lipid components of lipid rafts, and a specific subset (Brij98) of lipid rafts have been isolated from HIV-1 virions. Virion associated cholesterol and sphingomyelin have also been shown to be critical for HIV-1 replication, but the precise timing and mechanism remains unclear. A number of studies have investigated the contribution of different lipid classes to the HIV¬1 lipid composition, and recent advances in lipid mass spectrometry have allowed a more in depth analysis of the specific lipid species, not just classes, involved. This thesis provides the first study of the effects of viral genome, production method and producer cell on the phospholipid (specifically the lipid raft associated sphingomyelin and non-raft phosphatidylcholine) species distribution. These observations suggest that HIV-1 packages a relatively conserved subset of phospholipid species regardless of producer cell or viral genome. There is evidence to suggest that virion-associated sphingomyelin and phosphatidylcholine are important for HIV-1 infectivity. This thesis determined that sphingomyelin is required for both viral entry and infectivity. However, a reduction in virion associated phosphatidylcholine levels did not affect either entry or infectivity of HIV-1. These findings are in agreement with other studies which have shown that the lipid raft component sphingomyelin plays a role in the early stages of HIV-1 cell-cell fusion. While the stage of HIV-1 replication for which cell-derived cholesterol is required is currently undefined, it has generally been suggested that it is required for the very early stages of HIV-1 replication such as binding and fusion/entry into target cells. This thesis provides the first information that virion associated cholesterol is not required for HIV-1 viral entry but is required for a stage of replication prior to the completion of reverse transcription. In conclusion this thesis has demonstrated that virion-associated lipids play key, but different, roles in the early stages of HIV-1 infection. Data presented here show that the virion associated lipid (raft) components of HIV-1 are critical for both fusion/entry and for additional stage(s) post entry but prior to the completion of reverse transcription, possibly mediated through viral induced cellular signaling.