Studies on Herpes Simplex Virus Type 1 Latency in Tissue Culture Cells

Robinson, Laurence Henry (1996) Studies on Herpes Simplex Virus Type 1 Latency in Tissue Culture Cells. PhD thesis, University of Glasgow.

Abstract

After primary infection, herpes simplex virus type 1 (HSV-1) remains latent in neurons of the host. To facilitate studies on HSV-1 latency, models of latency have been developed in tissue culture. The HSV-1 protein Vmw65 is an essential structural component of the virus tegument. Following fusion of the virus envelope with the cell membrane Vmw65 is released into the cell and forms a complex with cellular factors which bind to TAATGARAT (where R is a purine) elements upstream of the viral immediate early (IE) genes. Once the complex has bound to DNA, the acidic carboxy-terminal domain of Vmw65 stimulates transcription by recruiting components of the RNA polymerase II transcription initiation complex to the promoter. The mutant ml814 has a4 amino acid insertion in Vmw65 which disrupts its ability to form the transactivation complex, as a consequence levels of IE transcription are reduced. During infection of cells with in1814 at low multiplicity of infection (MOI), only a minor proportion of infecting viruses undergo replication, whereas the majority of viral genomes enter a quiescent state. These quiescent genomes can be reactivated by expression within the cell of the HSV-1 encoded protein Vmwl 10 and probably by other herpesvirus proteins functionally equivalent to Vmw110. At high MOI in1814 replicates as efficiently as wild type virus. To perform structural and functional studies on the quiescent ml814 genomes, it is necessary to permit infection of cultured cells at high MOI. An in vitro latency system has been developed by D.R.S. Jamieson and C.M. Preston using two modifications which further reduce the expression of viral IE proteins, thereby enabling infection of human foetal lung (HFL) cells with in1814 at high MOI without extensive cell destruction. Firstly, the regulatory region controlling transcription of the gene encoding the IE protein Vmwl 10 has been replaced by the Moloney murine leukaemia virus (MMLV) enhancer. Since the MMLV enhancer is a relatively inefficient promoter in HFL cells the resulting mutant, named ml820, exhibits a phenotype equivalent to that of a Vmwl 10 deletion mutant of in1814 when titrated on HFL cells. Another modification is pretreatment of the cells with human interferon alpha (IFNalpha). Treatment of cells with IFNalpha results in an antiviral state against HSV-1 which has been attributed to reduced viral IE gene expression. Previous studies demonstrated that infection of IFNalpha-treated HFL cells with ml820 at high MOI results in high efficiency retention of viral genomes in a transcriptionally repressed state. The purpose of the study reported here was to extend previous analysis by D.R.S. Jamieson on the structure of the ml820 genome during latency in vitro, and to characterise early events in the establishment of latency. Initial experiments confirmed previous data showing that the ml820 genome is nonlinear in the in vitro latency system, as was reported to occur in neurons in vivo. Quantification of Southern blots revealed that after an initial input MOI of 120 particles of in820 per cell, approximately 84% of the total nuclear viral DNA was nonlinear and that on average each cell retained approximately 12 viral genomes. Upon reactivation of latent in820 by superinfection of latently infected HFL cells in the presence of an inhibitor of DNA replication, the reactivated genomes remained nonlinear, demonstrating that a change from a nonlinear to a linear form is not a requirement for reactivation. The configuration of the linear genomes also remained unchanged upon reactivation. During in vitro latency, the nonlinear viral genomes were more sensitive to nucleases than the linear genomes. Linearity and resistance to nucleases is indicative of genomes which are not uncoated and are thus unable to circularise. Comparison of rates of uncoating after infection of BHK, HeLa, CV-1 and HFL cells revealed that uncoating appeared significantly slower in HFL cells than in the other cell types tested. Latent HSV-1 DNA in the brainstems of mice is associated with nucleosomes in a chromatin structure. Previous studies examining the structure of in820 in the in vitro latency system showed that the thymidine kinaase (TK) gene is not bound by nucleosomes with regular spacing. The study reported here also showed the absence of a regular chromatin structure on the TK gene and furthermore that the nature of the inhibitor of DNA replication used to prevent the spread of nonlatent virus was not a significant variable. Fascinatingly, the LAT/Vmw110 encoding region, the only region transcriptionally active during latency in vivo, was in a regular chromatin structure in the in vitro latency system. However, when the LAT / Vmwl 10 encoding region and the TK gene were examined in a parallel experiment, both regions exhibited a possible regular nucleosomal structure suggesting that a regular nucleosomal structure exists on the entire genome and is not confined to the LAT/Vmw110 region. Thus the previous inability to detect chromatin on the TK gene may have been caused by differences in the arrangement of nucleosome between experiments or experimental differences in the ability to detect nucleosome-associated DNA. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Chris Preston
• Keywords: Virology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75768
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 18:13
• Last Modified: 19 Nov 2019 18:13
• URI: https://theses.gla.ac.uk/id/eprint/75768