Does HCMV have multiple escape routes?
Posted on October 6, 2023 by Dr Matthew Reeves
Dr Matthew Reeves takes us behind the scenes of their latest publication 'Repression of the major immediate early promoter of human cytomegalovirus allows transcription 2 from an alternate promoter’ published in Journal of General Virology.
My name is Matthew Reeves and I am a Group Leader at the Institute of Immunity & Transplantation which forms part of the Division of Infection & Immunity at UCL in the UK.
As a laboratory we are interested in viral pathogens that cause disease in vulnerable patient populations such as organ transplant patients. The pathogen of choice in the lab is human cytomegalovirus (HCMV), a member of the herpes virus family, although we are becoming interested in smaller DNA viruses like BK polyoma virus which also is a problem in our renal patients.
Going back to HCMV, a defining biological characteristic is the capacity of the virus to infect you for life and it is likely over 60% of the population are carrying the virus. Although our immune system cannot prevent HCMV infection (or even re-infection) we rarely see disease in individuals with a functional immune system. That said, the immune system devotes an impressive amount of resources to control this virus which likely explains why when we immune-suppress patients, the virus will cause disease. An important point, here, is that there is a view that immune-suppression, in itself, promotes reactivation - we caveat this and say that immune-suppression promotes clinical reactivation and resulting viraemia, not molecular reactivation, per se. At a molecular level we hypothesise viral reactivation is happening regularly but in healthy people this is controlled by the immune system.
Our research – along with our collaborators in Cambridge – seeks to understand the molecular mechanisms that control latency and reactivation with the view that we may be able to therapeutically intervene to target the latent virus – the holy grail of anti-virals directed against viruses capable of lifelong persistence.
Our most recent paper began as a systematic analysis of the action of drugs – specifically epigenetic modifiers – on HCMV gene expression. The rationale for this was that we showed many years ago that host chromatin regulates latency and reactivation. We also recently observed that inhibition of a family of enzymes, called histone acetyltransferases (HATs), could reduce viral reactivation by blocking the expression of the first viral genes required for reactivation (the IE genes) by controlling the activation of the major immediate early promoter (MIEP). Most investigators who work in eukaryotic cells will have worked with a variation of the MIEP as it is modified to create what is termed by many researchers as the “CMV promoter”, used in most plasmids to drive gene expression of genes of interest. Thus, we set out to test whether these same drugs worked in lytic infection. The phenotypes we were observing were modest and appeared to be bigger when the virus was present at lower multiplicities of infection (MOI). This MOI phenotype is something we have observed previously with other types of epigenetic modifiers.
In parallel to these studies, we were also investigating a recently reported observation from the Goodrum laboratory that the expression of the IE genes important for reactivation may be triggered by a unique promoter and not the MIEP. This challenged the long-held view that MIEP activity was the sole determinant of latency and reactivation. Indeed, I distinctly remember when the original report from the Moorman lab, that first identified these novel promoters/transcripts, came out that one of my first thoughts was also towards reactivation. What we and others are now appreciating is that it is all about context – both the MIEP and alternate promoters play a role depending on cell type and the way the cells are stimulated.
Returning to the HAT studies we were puzzled by an observation that some histone acetyltransferases inhibitors (HATi) did impact on IE RNA levels, but this just did not seem to translate into changes in protein. At this point Rebecca, a PhD student funded by the Royal Free charity leading these studies (and soon to be defending her thesis), decided to probe transcript identity in more detail. Rebecca realised that the effect of the HAT inhibitor was transient and, more importantly, when the MIEP was being inhibited, expression from the alternate promoter was easier to detect. This led us to analyse these observations in more detail in reactivation alongside lytic infection. What became clear was that the activity of these two promoters appeared interdependent and, specifically, it seems that when the MIEP is activated it predominates over other IE promoters. Is it possible that the virus has evolved a second mechanism which may allow it to reactivate when the MIEP cannot be de-silenced? This virus is one of the first to reveal itself in our transplant patients and this may be explained by the virus being able to reactivate through multiple mechanisms. This is one of the hypotheses we are looking to investigate in the future.
Moving forward, we would ultimately like to validate this in natural latency. All these data are based on robust and tractable experimental models that we and others have set up to study HCMV latency and we are now in the process of trying to determine whether reactivation of naturally latent virus follows the ‘same transcript rules’ as we have observed for experimental latency – in the context of IE transcript origin. The major issue we are dealing with is that the qPCR for detecting MIEP transcripts is much less sensitive than the qPCR that detects the alternate transcripts. Given the scarcity of IE transcripts in natural latency, we need to optimise the qPCR assays first to give us any chance of being able to do this effectively and objectively.
We consider this research important as it brings additional clarity to the regulation of HCMV IE gene expression during reactivation and highlights potential ways in which the virus has evolved to enhance its propensity to reactivate. It also provides us with a possible viral tool to start deciphering the mechanisms that are controlling this interdependent relationship between these two promoters and this could be important for understanding similar regulatory mechanisms active in eukaryotic cells to control host gene expression.