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insightLMU RESEARCH
Issue 04 · 2010
LIFE SCIENCES
SUSANNE WEDLICH
PROFILING
INTRUDERS TO PREVENT HOSTILE TAKEOVERS
Pathogenic viruses are an ever-present threat. Using human herpesviruses as his model,
virologist Professor Jürgen Haas has set out to elucidate systematically the complex molecular interactions that determine the outcome of a viral infection. A comprehensive
understanding of the interplay between host and viral functions should point the way
to more effective therapies for viral diseases.
A variety of evidence suggests that DNA was not always the central governor in living cells.
The ‘RNA World’ hypothesis posits that its close cousin RNA made the running in the early
evolution of life. RNA is known to have the capacity not only to store hereditary information,
but also to catalyze chemical reactions. It could therefore have mediated both functions in
the earliest viable cells – tasks which are now, in large measure, performed by other types
of molecules. Double-stranded DNA is chemically more stable than RNA, and is the almost
universal medium for genetic information storage in present-day organisms, while executive functions in modern cells are carried out by proteins. Owing to their almost limitless
structural variability, proteins can provide inert building material, reactive catalysts and
virtually everything in between. However, RNA still serves as the hereditary material in
many viruses, and plays essential roles at the interface between DNA and proteins in all
cells. RNA solves a logistical problem by acting as a go-between. Specific segments of the
DNA − the genes − encode the instructions for the amino acid sequences of proteins. In
multicellular organisms, the DNA resides in the nucleus, which is spatially separated from
the surrounding cytoplasm where proteins are synthesized. The gene sequences in the
DNA are first copied or ‘transcribed’ into single-stranded RNA molecules, which can cross
the nuclear membrane and enter the cytoplasm. There, these messenger RNAs (mRNAs)
program the synthesis of proteins (‘translation’) − provided they avoid an encounter with a
different class of RNAs, the so-called small, non-coding RNAs. There are several different
types of these sncRNAs, but all have one thing in common: they consist of no more than 30
subunits (nucleotides). This is much shorter than most mRNAs or other non-coding RNAs,
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such as the ribosomal RNAs that form part of ribosomes (120-4700 nucleotides long) − the
manufacturing plants for proteins in the cytoplasm − and the transfer RNAs (tRNAs, 80 nt)
which deliver the building blocks for proteins to the ribosomes. However, the sncRNAs play
important regulatory roles. Small interfering RNAs (siRNAs), for instance, bind to specific
mRNAs to form short double-stranded regions, inhibiting translation into protein and inducing their destruction. MicroRNAs (miRNAs) also regulate gene expression by binding to
mRNAs and modulating their translation in various ways. Individual miRNAs can probably
recognize several different species of mRNA.
SILENCING OF GENES
It is estimated that the expression of up to one-third of human genes may be regulated by
miRNAs. “This system serves to fine-tune the operation of many cellular functions, including
cell differentiation, cell division and cell death,” says Professor Jürgen Haas of the Max von
Pettenkofer Institute at LMU. “This implies that miRNAs play a key role in the development
of tissues – and perhaps also in the origins of cancers.“ siRNAs and miRNAs are a prime
subject of research worldwide, because they have great therapeutic potential. Indeed, the
phenomenon of RNA interference has already been exploited in experiments carried out in
cultured cells and in animal models. Artificial miRNAs have been designed and synthesized
and shown to inhibit the translation of the targeted mRNAs, which is equivalent to turning
off the corresponding gene. This effect could also provide a means of functionally inactivating specific genes that may induce pathological responses or are otherwise deleterious to
cells. Moreover, this is not the only conceivable application for small RNAs: they could also
be employed to prevent the subversion of cellular functions by viruses. Viruses are versatile parasites that cannot reproduce themselves independently. They must find and invade
appropriate cells, and then commandeer their resources to make new viral particles. The
simplest viruses consist of a nucleic acid (which may be DNA or RNA) and the coat proteins
that it encodes, which allow it to infiltrate into its unwitting host cells. Complex viruses have
more complicated genomes, and also make miRNAs, which regulate the expression of viral
genes or perturb the activity of host genes. The RNAs, unlike viral proteins exposed on the
surfaces of infected cells, are not recognized by the immune system. “The battle between
infectious agent and immune system begins as soon as the first cell has been infected,”
explains Jürgen Haas. “If the immune system identifies a cell as being infected, it normally
forces that cell to undergo programmed cell death, in a process called apoptosis. But many
viruses have evolved mechanisms that inhibit apoptosis.“
Some viruses induce their host cells to divide incessantly so as to maximize the production
of virus particles. Uncontrolled cell division is, however, a hallmark of malignant tumors,
and the development of such growths can be initiated by small numbers of viruses. Because
perhaps as many as 15% of human cancers can be attributed to their action, this group of
tumor viruses is of great medical significance. Two of the most important are Epstein-Barr
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Herpesvirus-host protein interaction network. In the diagrams, which were constructed with Cytoscape software, the points represent
proteins, while connecting lines indicate interactions. The panel on the left shows all viral proteins (green), the cell proteins with
which they interact directly (purple) and the latter’s normal partners (light blue). All other cellular proteins are included in the right
panel (gray).
Source: LMU Munich
Virus (EBV) and Kaposi’s Sarcoma-Associated Herpesvirus (KSHV). Both of these chronically infect the B cells of the human immune system. In the case of EBV, the primary infection usually occurs in childhood, and either provokes no obvious symptoms or induces a
harmless form of glandular fever. KSHV, also known as human herpesvirus Type 8, was first
identified in 1994, in patients with Kaposi’s sarcoma. In developed countries, this type of
skin tumor occurs mainly in AIDS patients whose immune defences have been weakened,
but Kaposi’s sarcoma is endemic in parts of Africa. The DNA genomes of herpesviruses
include sequences that are transcribed into miRNAs. Jürgen Haas set out to identify the
genes that are regulated by the miRNAs produced by EBV and KSHV. “This is a very young
research area,” he says. “Viral miRNAs were first described in 2004, and very little is known
about their regulatory targets. It is, however, clear that KSHV modulates the activity of
genes involved in the formation of blood vessels, in cell growth and in programmed cell
death.“ EBV miRNAs positively control genes that are required for cell survival. These findings provided the basis for a better understanding of the biology of these viruses. Detailed
knowledge of the molecular interactions between virus and host is, however, a prerequisite
for the development of novel therapeutic strategies for the treatment of such pathogens. Indepth cataloguing of viral miRNAs requires the use of elaborate, high-throughput genomic
and proteomic methods, but the rewards are worth the effort. “This approach has allowed
us to identify, with high specificity, over 100 target genes for viral miRNAs in six different
human B cell lines,” says Jürgen Haas.
The researchers were in fact able to pinpoint a total of 158 cellular genes that are targeted
by viral miRNAs. The miRNAs synthesized by EBV, for example, alter the metabolism of
the host cell in such a way that the immune response is compromised, and the infected
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cells fail to undergo apoptosis. “Using the same techniques, we also identified more than
2300 target genes for cellular miRNAs,” says Jürgen Haas. “The results provide an atlas of
the processes that are regulated by miRNAs in human B cells, and extend the list of known
targets fivefold.“ The significance of the work lies in the fact that the newly identified targets of miRNAs could potentially serve as points of attack for urgently needed new antiviral
agents. Indeed, it may be possible to turn the virus’ own weapons against it. miRNAs could
be specifically designed to bind to viral RNAs that are required for replication of the virus,
blocking their function and rendering them harmless. Jürgen Haas and his colleagues are
already hard at work. “Our aim is to define the key events in the pathogenesis of infectious
diseases based on genome-wide analyses of virus-host interactions, and then to characterize these in sufficient detail at the molecular level to allow the pharmaceutical development
of specific inhibitors.”
In order to attain this ambitious goal, his team is not only looking at the cross-connections
between viral miRNAs and cellular mRNAs, but also at interactions between viral and cellular proteins. Here too, they take advantage of various genetic and molecular biological
techniques, including RNA interference, which provide a genome-wide view of the interactions between infectious agent and host. When they discover a previously unknown virushost interaction, it is characterized in detail using biochemical, structural and functional
approaches. For Jürgen Haas, such analyses are necessary because − in the case of herpesviruses − viral gene products engage in hundreds of interactions. Instead of focusing
on specific components, this investigative strategy attempts to study systematically all viral
proteins and the host proteins to which they bind. The approach has proven so successful
that it has since been applied to an array of viral and bacterial pathogens, including influenza virus, the coronavirus responsible for SARS, and the bacterium Yersinia enterocolitica,
which causes diarrhoea. “We hope in this way to obtain a comprehensive picture of how
virus and host functions interact. Only when we thoroughly understand the interplay between them can we learn how to combat infectious agents effectively.“
Translated by Paul Hardy
Since 2006, Prof. Dr. Dr. Jürgen Haas has been Professor of Functional Genomics at the Chair of Virology in the
Max von Pettenkofer Institute at LMU Munich.
http://www.mvp.uni-muenchen.de/virology-workgroup-haas.html
http://www.bio.ifi.lmu.de/en/ngfn-plus
[email protected]
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