Maintaining the balance: persistent baculovirus infection in insect cells

Baculoviruses such as Autographa californica nucleopolyhedrovirus (AcMNPV) are insect-specific viruses that usually kill the host. However, many species harbour persistent, non-lethal covert baculovirus infections although these have rarely been observed in vitro. Hence, little is known about the mechanisms for establishment or maintenance of persistent infections. Re-infection with a homologous baculovirus (known as superinfection) has been shown to activate the persistent infection to an overt form. In vitro studies have shown that infected cells can be resistant to superinfection although the mechanisms involved are largely unknown. In 2011, a serendipitous event led to the creation of a persistently infected Trichoplusia ni cell line (Hi5) with an AcMNPV p10-deletion mutant (AcUW1.lacZ). From this culture, a clonal cell line (C20) was established that has been maintained to this day. This thesis aimed to use C20 to further our knowledge of the fine balance between host and virus (designated AcC20) that promotes the establishment and maintenance of a persistent infection. The cell line also enabled study of the superinfection exclusion phenomenon. C20 cells demonstrated remarkable viability and diversity of cell size/shape compared to the parental cells. The persistent AcC20 virus was maintained at low levels but budded virus (BV) titres varied considerably within and between different cultures. Presence of BV and the major membrane protein GP64 confirmed C20 harboured a persistent rather than latent infection. Analysis indicated that only a small proportion (<7%) of cells were producing BV whilst superinfection studies demonstrated that the majority of cells were resistant to a secondary infection. Microscopy studies indicated that superinfection may be blocked as early as the cell binding stage although virus uptake was also affected. Whole genome analysis of AcC20 showed four major deletions or insertions with other mutations in 34 coding regions affecting eight essential genes and a homologous region. Transcriptome analysis showed that all virus genes were expressed in C20 cells although the regulated phases of gene expression observed in a typical lytic infection were absent. Host cell transcriptome analysis also revealed differences between the cell response to a lytic and a persistent infection. In particular, endocytic pit formation and host defence pathways appeared to be compromised. The former may help explain resistance to superinfection and the latter may help maintain the persistent infection. Attempts to create further persistent cell lines proved difficult. However, using viruses with a mutation in lef2, in which BV production is severely reduced, overcame this problem. These persistent infections were also used to evaluate the possibility of continuous recombinant protein production by expressing dsred and urokinase under strong promoters. DsRed but not urokinase was produced over several cell passages. This thesis demonstrated establishment and maintenance of persistent infections in cell culture. Further research is needed to understand this process and why these cells are resistant to superinfection.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2019

Author dc:creator

• Garcia, Raquel Baptista Arinto

Contributors dc:contributor

• Possee, Robert
• King, Linda
• Hawes, Chris

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/5y02-x554

OAI identifier oai:identifier

tle:21b6bfa7-4800-4b89-99b3-8025644a8f1c:d6bd9758-527a-46cd-bfe2-c433766e8fca:1