Chivukula, Vidya; (2021) Probing the expanded architecture of the lentvirial intasome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Integration of viral DNA into a host chromosome, by action of the viral enzyme integrase (IN), is an essential step of the retroviral lifecycle. To fulfil its function, IN assembles into a multimer on the viral DNA ends, forming a highly stable nucleoprotein complex known as the intasome. The intasome architecture varies between the retroviral genera, and the maedi-visna virus (MVV) intasome contains a homo-hexadecamer (tetramer-of-tetramers) of IN. The conserved intasome core, observed in all structurally characterized retroviral intasomes, is formed between a pair of MVV IN tetramers, each providing one active site, and is completed by the insertions of the synaptic C-terminal domains (CTDs) donated by a pair of flanking IN tetramers. It was argued that this configuration is necessitated by the propensity of lentiviral INs to form tetramers in solution and the α-helical structure of the linkers connecting the catalytic core domain (CCD) and the CTD. Within the MVV IN hexadecamer, a pair of CTD tetrads bridge the IN tetramers by forming intra- and inter-tetramer interactions. Using site directed mutagenesis, the importance of these distinctive structural features was probed. The mutations disrupting the CTD-CTD interfaces or destabilizing the α-helical configuration of the CCD-CTD linkers perturb the ability of MVV IN to form multimers, assemble into stable intasomes and strongly affect its strand transfer activity in vitro. Moreover, these mutations strongly compromised infectivity of single-cycle MVV vector in cells. Lentiviral integration distinctively favours actively transcribing genes, which is facilitated by the interaction of IN with LEDGF/p75, a chromatin-bound adaptor protein. The presence of LEDGF/p75 was essential to observe MVV IN strand transfer activity or intasome assembly in vitro. To determine the importance of LEDGF/p75 for integration in the context of viral integration in cells, infectivity of MVV-derived vector in LEDGF-knockout cells was tested. Although ablation of the host factor in human and sheep cells did not lead to a reproducible reduction of infectivity, it led to a notable shift in integration pattern, away from the usual gene bodies and transcription units. Collectively, these observations indicate that the hexadecameric architecture is critical for the MVV IN function and suggest that LEDGF/p75 is relevant in targeting its integration towards favored regions of host genome.

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