Studies Using Pseudotyped Retroviral Vectors

Mahoney, Catherine H (1999) Studies Using Pseudotyped Retroviral Vectors. PhD thesis, University of Glasgow.

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The host range of a retrovirus depends largely upon the properties of the envelope glycoprotein, Env (Hunter, 1997). As a result this host range can be changed by replacing the envelope protein with that from another virus. The pseudotypes formed in this process possess the host range and fusion properties of the virus donating the envelope protein (Zavada, 1982). As retroviruses integrate into the genome of the infected host cell they have proved useful for the development of vectors for gene delivery (Miller, 1997). The host ranges of these vectors are restricted to cells that express the specific receptor recognised by the particular envelope protein. Several pseudotyped vectors have therefore been developed in order to increase host range or to target the vectors to specific cell types. The production of retroviral vectors pseudotyped with the envelope glycoprotein of the rhabdovirus vesicular stomatitis virus (VSV) was a significant advance in this field. Not only did these vectors display the wide host range of VSV, but they were also very stable, allowing concentration to high titres. Unfortunately the VSV envelope protein causes cell fusion when expressed at high levels, making continual production of these vectors very difficult. In this study an attempt was made to produce similar vectors pseudotyped with the envelope glycoprotein of another rhabdovims, rabies vims. These vectors displayed the host range properties of rabies, but they were produced at very low titres. The glycoprotein was capable of inducing cell fusion at low pH, indicating that it was functional. Therefore the low titres suggest that the rabies envelope protein was not incorporated efficiently into the retroviral vectors. Although these pseudotyped vectors did not prove to be useful as gene delivery vectors, the observations described here may provide interesting information on the processes of virion assembly and envelope incorporation. The second part of this study involved investigations into the infectivity of the porcine endogenous retroviruses. Concern has been raised recently over the potential infectivity of these viruses following proposals to use porcine organs and tissues in human transplants. Endogenous retro viruses are integrated into the host germ line (Boeke and Stoye, 1997) and would therefore be present in all transplanted cells. Initial cocultivation experiments indicated that these viruses could infect human cells. No obvious interference was observed with other mammalian C-type retroviruses, indicating that the porcine viruses recognise a novel receptor. Subsequent sequence analysis has revealed that at least three viral subgroups exist in the porcine genome. The determination of the Env sequence of one of these viruses is reported here. The encoded protein has a very similar structure to the Env proteins of other mammalian C-type retroviruses, but shows significant differences in the predicted receptor binding regions. The porcine virus subgroups also differ from one another within these regions, suggesting that these viruses recognise different receptors, and may therefore have different host ranges. Another potential application of the ability of retroviral vectors to incorporate foreign envelope proteins is in studies of the infectivity and host range of new viruses. In order to characterise these properties of the porcine endogenous retroviruses, pseudotyped vectors were produced, bearing the Env proteins of the three subgroups. Infected cells were then simply identified from the expression of a marker gene inserted in the vector genome. Analysis of the vectors revealed that at least two of the subgroups can infect human cells, and could therefore present a risk of infection to transplant recipients. Consequently it is important that further studies of these viruses are carried out in order to assess their tissue tropism and potential pathogenicity. These pseudotyped vectors should also be useful for such investigations of Env function and receptor usage of the porcine endogenous retroviruses.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David Onions
Keywords: Virology
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-75960
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:12
Last Modified: 19 Nov 2019 17:12

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