Research Article: The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

Date Published: April 7, 2016

Publisher: Public Library of Science

Author(s): Michael P. Nicoll, William Hann, Maitreyi Shivkumar, Laura E. R. Harman, Viv Connor, Heather M. Coleman, João T. Proença, Stacey Efstathiou, Lindsey Hutt-Fletcher.

http://doi.org/10.1371/journal.ppat.1005539

Abstract

Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir.

Partial Text

Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are ancient human pathogens that are most commonly associated with sub-clinical and mild infections but can occasionally cause severe life threatening disease [1]. HSV-1 is most commonly associated with infection of the oral mucosa, and following productive primary infection at this site the virus is able to access the sensory neurons of the trigeminal ganglia (TG). Within these cells, HSV is able to establish a latent infection, characterised by a global reduction of lytic gene expression and an absence of infectious virus production. Periodically, latency is interrupted by reactivation of virion production from latent viral DNA, allowing for the transmission of the virus to new hosts. During latency, viral gene expression is largely restricted to the latency-associated transcript (LAT). The LAT is an 8.3kb primary transcript, which is spliced into stable 1.5 and 2 kb major LAT introns, as well as a 6.3 kb minor LAT exon that is processed into a number of microRNAs. The HSV LAT and its associated microRNA species appear to limit HSV immediate-early (IE) gene expression in vitro [2–4], as well as limiting the accumulation of viral lytic gene transcripts during acute and latent infection of mouse models [5, 6]. The LAT intron has also been strongly implicated in the global control of latent HSV gene expression in a number of studies describing the post-translational modification (PTM) of histones associated with viral promoters [7–11].

In this study we have generated luciferase and β-galactosidase-expressing HSV-1 recombinants in order to examine reporter gene expression at the level of both whole ganglia and single cells. We have observed that LAT-negative virus infections express higher levels of reporter gene during latency in TG, and this is probably the result of an increased number of infected cells in which virus gene expression occurs at any one time. Furthermore, by isolating individual latently infected cells and culturing ex vivo, this heightened gene expression is mirrored by a detectable increase in reactivation at the single cell level. Whilst we cannot formally rule out down-regulation of both firefly luciferase and β-galactosidase reporter genes by HSV-1 microRNAs, we propose that HSV-1 LAT expression limits the rate of virus reactivation, and does so by restricting gene expression from the latent genome.

 

Source:

http://doi.org/10.1371/journal.ppat.1005539

 

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