DR NICOLA ROYLE
Testing telomeres
Dr Nicola Royle is investigating telomeres and their relation
to ubiquitous herpesviruses in humans. Here, she discusses the
inspiration behind her work to unravel viral latency and reactivation
many rounds of DNA replication from
developing into a tumour. However, there is
a downside: the accumulation of senescent
cells compromises tissue function and
contributes to the ageing process. Thus,
telomeres play various roles in cancer
and ageing.
How did you become interested in
human herpesviruses (HHVs)?
Could you introduce your research and
explain why it excites you?
I find telomere molecular biology
absolutely fascinating! Telomeres are
DNA-protein complexes that form
essential protective caps at the ends
of chromosomes, yet are themselves
inherently unstable. This puzzle has kept
me interested in them for many years.
When telomeres are present and functional,
the 23 pairs of chromosomes in a human
cell are stable throughout the cell cycle.
However, failure of the capping function
results in abnormal chromosomes that
break during cell division, causing genome
instability and leading to cancer.
Even more intriguingly, telomeric DNA
sequences are lost at every cell division,
resulting in a shortening rate in humans
of about 50 base pairs per year. The
gradual erosion of telomeres eventually
triggers cellular senescence, and the cell
stops dividing. Cell senescence has most
likely evolved as a protective measure
against cancer, as it prevents cells that
may have acquired mutations through
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INTERNATIONAL INNOVATION
My interest in HHVs, specifically in the two
species of human herpesvirus-6 (HHV-6A
and HHV-6B), started through a chance
meeting with my colleague Professor
Martin Dyer at a workshop. Dyer told me
about a patient he had treated for a type of
lymphoma that is normally triggered by a
virus. The patient turned out to be carrying
a chromosomally integrated copy of the
human herpesvirus-6 (CI-HHV-6) in one
telomere. What could be more fascinating
than a herpesvirus that can insert its DNA
into a telomere? I jumped at the chance
to collaborate.
About 1 per cent of the world’s population
is a CI-HHV-6 carrier, with a single copy
of HHV-6 integrated into one telomere
that they inherit from a parent and can
then pass onto their children. Much of the
interest in CI-HHV-6 to date has focused on
the effect that telomeric integration has on
HHV-6 as a viral pathogen. For example, is
integration an evolutionary dead-end or a
novel form of latency? These are fascinating
questions, but I am also interested in what
impact the insertion of a viral genome has
on the telomere.
Do other herpesviruses behave in the
same way?
No other HHVs are known to integrate into
telomeres, although HHV-7 does contain
telomere-like repeats. Interestingly though,
Marek’s Disease Virus (MDV), a related
herpesvirus that infects chickens, becomes
latent and upon reactivation, it causes
T-cell lymphomas. Unvaccinated birds are
often infected at a young age and latency
is established via telomeric integration.
Viral reactivation generally occurs as a hen
approaches the age at which it lays eggs,
causing multiple lymphomas that can
quickly kill the bird. Vaccines against MDV
are in use, but there is increasing evidence
that vaccinated birds are not resistant to
newly emerging strains. It is likely that we
could adapt the assays we have developed
to study HHV-6 to investigate MDV
integration, latency and reactivation with
a view to learning how these processes
are controlled.
Why is polymerase chain reaction
(PCR) such a valuable technology for
your purposes? How is it expediting the
discovery process?
My group has considerable expertise in
the analysis of human telomeric DNA,
because we have developed a variety of
PCR-based methods to isolate telomeric
sequences from individual chromosomes
and characterise the processes that cause
mutations within the repetitive DNA. I
DR NICOLA ROYLE
Viral integration
Researchers from the University of Leicester are investigating
the connection of human herpesvirus-6 with telomeres. Their
work is advancing the understanding of fundamental genetics
as well as the release of a potentially pernicious virus
TELOMERES ARE TANTALISING structures that
hold some of the secrets to ageing and cancer. These
unique entities comprise a repeated sequence of
six nucleotides and are found at the end of linear
chromosomes. Without telomeres, the free ends
of DNA molecules cause problems in eukaryotic
cells, as they can fuse together. If a cell with fused
chromosomes replicates, the DNA molecule breaks
and important genetic information is lost. Telomeres
act as caps to prevent this; scientists have compared
them to the aglets on the ends of shoelaces.
soon realised that, because most of the
HHV-6 genome is single copy sequence and
distinct from other sequences in the human
genome, we could develop PCR-based
assays specifically to investigate CI-HHV-6.
We have used these assays to study
integrated viral genomes and measure the
length of the telomere associated with the
HHV-6 integration, which is often short.
We have shown that terminal cleavage and
packaging sequences (PAC1 and PAC2) are
lost from CI-HHV-6 during integration.
Therefore, the integrated virus does not
have a full-length direct repeat (DR) region
at either end of its genome.
Could you discuss some of your most
significant discoveries?
We have evidence that the HHV-6 genome
can be released from the telomere. The
released viral DNA contains a single, fully
reconstituted DR, with both packaging
sequences. This DR could only arise through
a recombination event leading to release
of the entire viral genome as a circular
molecule containing a single, complete DR.
This is the likely first step towards rolling
circle replication and viral reactivation.
Telomeres play an important role in health. For
example, as we age, telomeres themselves become
shorter, which signals the cell to stop dividing. This
process is called cellular senescence. Telomeres
also prevent chromosomes fusing or rearranging,
activities that would otherwise lead to cancer.
These crucial structures fascinate Dr Nicole Royle,
Senior Lecturer in the Department of Genetics at
the University of Leicester, UK. She has extensive
knowledge about human telomeres and expertise
in their analysis, both of which she is currently
applying to the herpesviruses.
LAYING DORMANT
Herpesviruses are a large family of DNA viruses.
There are nine known to cause disease in humans,
called the human herpesviruses (HHVs). Royle is
particularly interested in the two HHV-6 species, A
and B, which infect almost all human populations.
They display the characteristic behaviour of
herpesviruses – they lie dormant for years following
infection. In this latent state, the virus persists in low
numbers, is asymptomatic and can endure in some
cells for a lifetime.
However, when reactivated, HHV-6 can cause
serious harm and has been associated with
drug induced hypersensitivity syndrome. The
consequences of reactivation range from mild to
severe in immunocompromised individuals, such
as transplant patients, because HHV-6 reactivation
can cause health issues such as a fever and rash
through to seizures, cognitive dysfunction and
encephalitis. Reactivation has also been associated
with delayed engraftment.
The latent form of most HHVs is a circular,
independent structure called an episome. Given
the opportunity, both HHV-6A and HHV-6B can
integrate into telomeres, because the genome
contains two identical terminal regions that contain
the same repeat DNA sequences as telomeres.
This aspect allows them to insert into the
telomeric repeat array to become chromosomallyintegrated (CI-HHV-6). Almost 1 per cent of the
UK population – over 500,000 people – are carriers
of this integrated form. Recently, the integratedvirus has been shown to reactivate under certain
circumstances. The potential adverse effect of CIHHV-6 on telomere function and length regulation
– processes with hugely important consequences –
has hardly been explored.
Telomeric integration has been characterised in
individuals who inherit CI-HHV-6 from a parent,
but the potential for HHV-6 integration in somatic
cells in the majority of the population following a
childhood infection, has not been explored. This
raises an interesting question for Royle’s research:
is HHV-6 latency in somatic cells via an episomal
form, via telomeric integration or even a mix
of the two? “The consequences of the different
forms of latency may vary, as reactivation of the
integrated form may also effect telomere function,”
she explains.
VIRAL MODIFICATION
In order to answer fundamental questions, Royle’s
team have conducted a range of investigations.
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INTELLIGENCE
HHV-6, TELOMERES AND
CHROMOSOME STABILITY
OBJECTIVES
• To investigate cellular processes that
affect the stability of telomeric DNA
in cells and during cancer initiation
and progression
• To understand how HHV-6 latency
following telomeric integration affects
telomere function, viral release,
reactivation and evolution
KEY COLLABORATORS
Professor Martin Dyer, University of
Leicester, UK • Dr Duncan Clark, Barts
Health NHS Trust, UK • Professor Ruth
Jarrett; Dr Andrew Davison, University of
Glasgow, UK • Dr Ursula Gompels, London
School of Hygiene & Tropical Medicine, UK
FUNDING
Medical Research Council (MRC) •
Wellcome Trust Institutional Strategic
Support Fund • University of Leicester •
Cancer Research UK
ACKNOWLEDGEMENT
All members of the telomere research
group for their contributions to the project,
in particular Ms Enjie Zhang and Drs Yan
Huang and Victoria Cotton.
CONTACT
Dr Nicola Royle
Senior Lecturer
Department of Genetics
University of Leicester
University Road
Leicester
LE1 7RH
UK
T +44 116 252 2270
E [email protected]
www2.le.ac.uk/departments/genetics/
people/royle
NICOLA ROYLE graduated with a BSc in
Genetics and Cell Biology from the University
of Manchester, UK, and received her PhD
in Genetics from University of Reading,
UK. Following six years as an MRC Human
Genome Mapping Project Senior Fellow, she
joined the academic staff in Department
of Genetics at the University of Leicester,
where she currently holds the position of
Senior Lecturer. She is focusing her research
efforts on cellular processes that impact on
the length and stability of telomeric DNA in
somatic cells and in the germline.
Firstly, to understand the frequency of CI-HHV-6
in the worldwide population, the team screened
for HHV-6 sequences in DNA samples from various
populations, including the international HapMap
collection. They confirmed that 1 per cent of
people worldwide are CI-HHV-6 carriers. They also
identified over 70 individual carriers, and obtained
their cell lines and DNA samples.
Interestingly, when the team analysed the
chromosome-virus junctions, they found that the
integrated virus is not the same as its independent
form. Two important sequences were missing from
the genome – PAC1 and PAC2, the packaging and
cleavage sequences. However, one copy of each
is retained at an internal location, preserving the
possibility for reactivation.
The team also analysed viral gene expression, with
findings indicative of a latency mechanism. They
consistently found expression of the U90 gene,
which plays an important role in controlling latency.
TRUNCATED TELOMERES
After clarifying the effects on the virus, Royle
moved on to study the effect of integration on the
telomere. Her team compared the length of the
telomere on the end of the virus to other telomeres
in the somatic cells of CI-HHV-6 carriers. The
findings were surprising: the CI-HHV-6-associated
telomeres were often the shortest measured.
However, the virus-associated telomere was not
the shortest found in sperm DNA, showing that
telomerase, the enzyme responsible for extending
telomeres, is able to lengthen the telomere in
the germline. This led Royle to question how the
virus-associated telomere becomes so short in
somatic cells.
The answer to this might lie in t-loops, the
capping structures telomeres form. “Telomeres
are composed of double-stranded DNA, but are
terminated by a single-stranded extension. The
double-stranded portion is bent into a looped
structure that facilitates interaction with the
terminal, single stranded overhang. This t-loop,
stabilised by the Shelterin complex, caps the end of
the chromosome,” Royle explains. These loops can
be excised to release a t-circle, a circular molecule
containing telomeric DNA, and evidence suggests
this process is used as a ‘trimming’ mechanism to
curb lengthy telomeres.
VIRAL RELEASE
Building on this, Royle suggested that t-loops could
actually be formed at the virus-associated telomere,
including the viral genome. In fact, the team found
extra-chromosomal molecules containing viral
DNA, suggesting that the telomere releases the viral
genome via a t-loop intermediate.
A t-loop containing part, or indeed all, of the viral
genome would fail to bind the Shelterin complex,
and thus the telomere’s capping function would
be impaired. This chimeric t-loop structure could
be processed, leading to the release of circular
viral molecules – the first step of reactivation.
Royle’s team has provided robust evidence that
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INTERNATIONAL INNOVATION
ASSAY
DEVELOPMENT
Royle’s research could become the basis of a
kit to detect and quantify HHV-6 telomeric
integration in somatic cells. This could be
used to assess transplant organ donors
or recipients before treatment, so viral
reactivation can be predicted and treated,
improving long-term outcome.
CI-HHV-6 may have unknown long-term
health consequences, so screening sperm
donors for the integration may become a
useful tool.
the integrated virus does in fact affect telomere
function and suggests that viral excision, which
likely precedes reactivation, depends on the
formation of t-loops.
Through her many and varied investigations,
Royle has made several important findings. Taken
together, they clearly show that viral integration
affects the length and stability of the associated
telomere, thereby aiding the release of circular
viral molecules, some of which could become fully
functioning viruses. Royle’s model, which states
that partial or complete excision of integrated
viral sequences takes place via the processing of
chimeric t-loops at telomeres, is completely new
and will change understanding of both telomere and
herpesvirus biology.
EXPLORING THE UNKNOWN
Although she has made significant progress, Royle
has many unanswered questions in this largely
unexplored area. For example, the frequency of
different germline integration events remains a
mystery. She is addressing this by sequencing HHV6 genomes from unrelated CI-HHV-6 carriers. She
will compare the sequences with the aim of finding
distinctive features in those strains that integrate in
the germline.
The team will also further investigate telomere length
dynamics. If extensive trimming in the germline
can excise the viral genome from a telomere, this
would have important ramifications for gamete
production and virus reactivation in carriers and
their offspring. Royle also plans to identify factors
that affect the release of the viral genome and
subsequent reactivation. “It is important to know
if drugs that affect chromatin organisation, such as
histone deacetylase (HDAC) inhibitors, affect HHV6 reactivation,” she concludes.
Detection of HHV-6 (green) at a telomere of one copy of
chromosome 11 (blue) from an individual with CI-HHV-6B.