Regulation of late and very late gene expression in both lytic and persistent baculovirus infections

Autographa californica nucleopolyhedrovirus (AcMNPV) is an insect-specific pathogen, with a biphasic replication cycle generating both budded viruses (BVs) and occlusion bodies (OBs). While BVs are released from the cell membrane mainly in the late phase of infection, OBs are produced in the subsequent very late phase and released after cell lysis. A characteristic of this very late phase of infection is the hyperexpression of the viral genes p10 and polyhedrin (polh), which are nonessential in vitro. While polyhedrin is a highly robust protein accumulating in the nucleus of virus-infected cells surrounding the virions and offering a degree of protection, the precise role of P10 is still unclear. It appears to have various roles at the end of the viral replication cycle such as nuclear stability and nuclear disintegration. Importantly, this stage has been utilised to produce recombinant proteins (e.g. vaccines). However, persistent baculovirus infections are also known. They are maintained throughout the life cycle of the host without causing mortality and insect liquefaction for polyhedra dissemination. The molecular mechanism of how the fine balance of host and virus gene expression is maintained remains elusive. Therefore, the aim of this thesis was to determine how late and very late genes in both lytic and persistent baculovirus infections are regulated. To investigate the regulation of AcMNPV gene expression the effect of the candidate genes cathepsin, chitinase A, late expression factor 2 (lef-2) and vp39 were analysed on very late gene expression in overt infections by various approaches such as gene knock-outs, mutagenesis studies or RNA interference. The late proteins cathepsin and chitinase A are viral enzymes required for liquefaction of the viral host in vivo. Moreover, cathepsin was implicated to be involved in cell lysis. It could be determined that P10 was specifically cleaved by the cathepsin independently of chitinase A. Moreover, it appears that processed P10 was crucial for nuclear disintegration and polyhedra release. Lef-2 is an early viral gene required for AcMNPV DNA replication. Additionally, it was previously hypothesised that it may be important for very late gene expression. Mutagenesis studies on LEF-2 confirmed that different protein domains are crucial during DNA replication and BV production, while its role in very late gene expression remains elusive. VP39 is a structural protein and the major capsid protein in AcMNPV. However, it was also speculated that it may be involved in p10 and polh expression. Knock-down of vp39 and subsequent analysis of the effect on mRNA abundances confirmed not only the down regulation of very late gene expression but showed moreover a global effect on viral gene transcription. Additionally, a more global approach by RNA-sequencing was performed to compare host and virus gene expression in both lytic and persistent AcMNPV infections utilising an in vitro system, the Trichoplusia ni C20 cell line that supports the continuous replication of low-level infection of AcMNPV lacking p10. It could be determined that all AcMNPV genes were expressed in C20 cells at low levels but without distinct temporal phases or shut down of host gene expression. Additionally, transcription of genes required for specific cellular pathways such as endocytosis were altered compared to AcMNPV-infected T. ni Hi5 cells. Recent investigations to recreate an insect cell line harbouring a persistent baculovirus were unsuccessful. It was proposed that improved BV production was too high to allow a persistent state of virus replication to be achieved. Hence, a virus with an impaired BV production, AcLEF-2C84A, was utilised to generate a new T. ni Hi5 cell line containing a persistent baculovirus. Preliminary characterisation of this cell line (AcLEF-2C84A/Hi5) revealed similarities to the C20 cell line with respect to its resilience and continuous production of BV.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2018

Author dc:creator

• Bannach, Carina

Contributors dc:contributor

• King, Linda
• Possee, Bob

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/a8fx-yc97

OAI identifier oai:identifier

tle:d921555d-b914-4c3a-8967-d81d23d57d98:d6bd9758-527a-46cd-bfe2-c433766e8fca:1