The unusually long Cytoplasmic tail (CT) of Human Immunodeficiency Virus Type-1 (HIV-1) glycoprotein-41 (gp41) is highly conserved and engineered large truncations often render the virus non-infectious in a cell-type dependent manner. While large CT truncations occur infrequently in natural isolates, little is known about the mechanisms involved in infectious virions harbouring a large CT truncation. This thesis characterises RFgp34, a replication competent laboratory HIV-1 isolate with an acquired 100 amino acid CT truncation, and how it diverged from wildtype RF. The CT truncation and two possible compensatory mutations in Matrix (E40K and F44I) were introduced into the HIV-1 isolate NL4-3. These mutants were tested for infectivity, syncytia formation and glycoprotein incorporation into virions, alternative co-receptor usage and sensitivity to the fusion inhibitor T-20. Compared with RFwt, RFgp34-infected cultures displayed delayed viral replication kinetics in all cell types. Similar sized (MT-4 cells, PBMC) or larger and more numerous syncytia (Hut78 cells) were detected in RFgp34-infected cultures. Similar (Hut78 cells) or decreased (MT-4 cells, PBMC) amounts of glycoprotein was incorporated into RFgp34 virions, compared with RFwt virions. The increased syncytia in RFgp34-infected Hut78 cultures and the reduced glycoprotein incorporation into RFgp34 virions from MT-4 cells and PBMCs may explain the delayed RFgp34 replication kinetics. The Matrix E40K and F44I mutations were not able to directly compensate for the CT truncation to restore infectivity in Hut78 and MT-4 cells, as secondary mutations or the reversion of the CT truncation to a full-length CT were observed. In PBMCs the Matrix mutations alone were able to partially restore infectivity, suggesting specific mutations may compensate for the CT truncation in different cell types. None of the viruses utilised alternative HIV-1 co-receptors, nor were more resistant to T-20 than wildtype HIV-1 suggesting that the CT does not directly play a role in these viral functions. This thesis suggests that the sequence of mutations acquired by RFgp34 to compensate for the CT truncation and restore infectivity in multiple cell types may have occurred in a specific order and the evolution of RFgp34 to out-compete RFwt occurred over many passages.