Johnson, S; (2014) Comparative analysis of cellular proteins in stably and transiently produced lentiviral vectors. Doctoral thesis , UCL (University College London).

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Abstract

Lentiviral vectors (LVs) are successfully used in clinical trials showing long term therapeutic benefits. Studying the role of cellular proteins during replication of lentivirus HIV-1 helped to understand virus assembly and budding. Knowing what cellular proteins interact with viral proteins and identifying interactions that promote formation of functional particles can be valuable for improving LV production in Gene Therapy. The cellular protein composition of LVs produced by two different methods was compared, the transient transfection system producing vectors pseudotyped with the VSV-G envelope and a stable producer cell system producing vectors pseudotyped with the non-toxic retroviral envelope, RDpro. Lentiviral vectors were purified using size exclusion chromatography (SEC). The number of purified LVs produced by transient transfection was six fold higher compared to stably produced particles. For linear ion trap-orbitrap tandem mass spectrometry (LTQ-MS/MS) a comparable amount of SEC purified LVs was analysed, detecting a smaller number of cellular protein species in stably compared to transiently produced vector samples. The greater numbers of host proteins in purified transiently produced samples may due to the presence of co-purified VSV-G vesicles. On the other hand, a large number of proteins we identified had also been detected in studies of wild type viruses and HIV-1 derived vectors indicating a role in vector formation, such as viral protein transport to the assembly site in producer cells. The potential role in LV particle production of selected identified proteins was assessed. Whilst some proteins that have been detected in studies on wild type HIV-1 were found in all our samples, such as ALIX, AHNAK was unique to stably produced, RDpro pseudotyped vector samples, and thus selected for further investigation. In summary, knock down of ALIX, AHNAK and TSG101 host cell proteins in vector producer cells did not result in a significant difference in vector production.

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