Wight, DJ; (2013) Investigations into the function of the gammaretroviral protein p12 during the early stages of infection. Doctoral thesis (PhD), UCL (University College London).

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Abstract

The retroviral structural proteins (encoded by gag) are not merely the architecture of the virus but carry out a diverse set of functions throughout the viral life cycle. Initially translated as a polyprotein, the Gag protein functions to encapsidate the genome and assemble retroviral particles at the budding sites. This polyprotein is then cleaved into the mature proteins: matrix (MA), capsid (CA) and nucleocapsid (NC) upon viral release. Most retroviruses also contain a small protein between MA and CA of unknown function. In Moloney murine leukaemia virus (Mo-MLV) this protein is p12. p12 contains the late-domain, required for recruiting ESCRT machinery to promote virus budding, but also contains regions that have been shown to be important during the early stages of infection The aim of my PhD project was to establish the function of p12 during the early stages of infection and, in doing so, shed some light on the enigmatic processes occurring during these stages. Initially, the phenotypes of Mo-MLV virus-like particles (VLPs) containing previously identified p12 mutations were further characterised. Importantly, we found that these p12 mutations were clustered into two domains, both of which are required for Mo-MLV infectivity. Using genetic approaches, the order in which these domains act during infection was established. Furthermore, individual alanine mutants highlighted the residues within these domains that are essential for Mo-MLV infectivity. The conservation of p12 function was analysed by creating analogous p12 mutations in other gammaretroviruses. While the whole C-terminal domain was essential, some viruses contained dispensable portions of the N-terminal domain. Chimeric viruses were constructed between Mo-MLV and Gibbon ape leukemia virus (GaLV) to further assess the conservation of p12 function. Using biochemical, genetic and microscopic approaches, potential functions have been ascribed for the N- and C-terminal domains, allowing a model to be postulated for p12 function during the early stages of infection. This model proposes that the N-terminal domain of p12 binds to a virus specific factor which ties it to the pre-integration complex (PIC) and also stabilises the CA core. The C-terminal domain then comes into play during mitosis, tethering the PIC to the chromosomes and aiding integration. Therefore, the last gammaretroviral Gag protein to be given a functional name has a large role to play in replication. Further understanding of p12, building on the work presented in this thesis, could aid in the design of gene transfer vectors with more precise integration targeting, and potentially will lead to improved understanding of early replication events that could enhance therapeutics against more complex retroviruses that cause human disease.

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