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A virus that hides while it waits for an opportunity to replicate

Thomas Mertens, Medical Director of the Institute of Virology in Ulm, has a strong scientific and clinical interest in the human cytomegalovirus (HCMV), a big virus with a big impact which, despite its size, is overshadowed by known viruses such as the HI virus that is the object of research for Mertens’ colleagues. HCMV research is a challenging area where quick successes are rare.

A giant of virus - the HCMV list of threats is impressive:
The human cytomegalovirus, which belongs to the herpes virus family, is among the largest of human viruses and is widely distributed across the entire globe. The HCMV genome codes for more than 200 genes. Next to HCMV, hepatitis B viruses are dwarfs; their genome contains only two thousand or so nucleotides, while the HCMV genome has 200 times as many. The replication of the virus, which is transferred through body fluids, requires an ordered cascade of gene expression.

Temporally regulated gene expression

Prof. Dr. Thomas Mertens. CMV and influenza virus expert. © University Hospital Ulm

Mertens says that this is because the virus has a large number of genes that are not expressed simultaneously. The HCMV proteins are expressed in three temporal cascades; the virus encodes a large number of genes that are expressed in a temporally regulated manner. This starts with the expression of the immediate early genes (which have a regulatory function), is followed by the expression of early genes (those with enzymatic function such as replication enzymes for the viral DNA), and then the expression of the late genes, which encode for structural components of the viral particle.

The replication of the virus takes a relatively long time, which, according to Mertens, has something to do with the age of the virus. Evidence suggests that HCMV is an extremely old virus that has co-evolved with humans, with the result that people in good immunological health rarely suffer from HCMV infections due to the virus' adaptation to its host.

The schematic, which shows the different infectious stages of the human cytomegalovirus, reveals the complexity and diversity of interactions with infected cells and the different diagnostic and therapeutic possibilities available.
The schematic, which shows the different infectious stages of the human cytomegalovirus, reveals the complexity and diversity of interactions with infected cells and the different diagnostic and therapeutic possibilities available. © Mertens

Complex and crafty

Because of the size of the HCMV genome, the researchers need to look at a large number of interactions between the human cells and the viruses. Another problem for the researchers is that, although the virus remains dormant in the body following primary HCMV infections, it can be reactivated. Thomas Mertens explains that although many people carry the virus, they remain healthy as long as they have an effective immune system. The virologist also explains that one person in two in the Ulm region carries the virus; in other places around the globe, where hygiene standards are lower, almost everyone carries the virus.

It is now known that the virus retreats into specific body cells known as the monocytes and probably also into endothelial cells. But Mertens points out that not all virus hiding places are known. The dormant virus can be reactivated through a variety of different mechanisms.

Risk for people with a weak immune system and for unborn babies

According to Mertens, HCMV is the most common pathogen known to infect unborn babies as a result of their mothers becoming infected with HCMV during pregnancy. In addition, the virus is highly pathogenic, sometimes even fatal, for transplant and cancer patients with a weakened immune system in whom it leads to pneumonias or gastrointestinal ulcerations. According to Mertens, one in three people with a bone marrow transplant are at risk of contracting HCMV infections. Besides fungi and bacteria, HCMV is the most important pathogen to put transplanted patients at risk.

When and how are latent viruses reactivated?

It is still not known in detail why the virus is frequently reactivated in pregnant women. Mertens believes that molecular trigger mechanisms are present. It would appear that the immune system of HCMV-infected people is key in virus reactivation, since an active virus infection is mainly controlled by CD8-positive (cytotoxic) T-cells. It is also clear that latent viruses only express a small number of their genes. Little is so far known about latency and about reactivation genes.

When the latent virus is reactivated, it starts to replicate in cascades, which is due to the temporally regulated expression of the large number of genes involved.
When the latent virus is reactivated, it starts to replicate in cascades, which is due to the temporally regulated expression of the large number of genes involved. © Mertens

Rapid infection, slow replication

The replication of viral DNA occurs in the nucleus of the infected cells via the rolling circle mechanism, which produces multiple copies of a single circular template. The viral DNA is joined to a ring, and is subsequently cleaved into DNA fragments of a specific length, which are packed into capsids and released into the extracellular space across the inner and outer cell membrane by way of the Golgi membrane. Whilst HCMV viruses can quickly infect cells, their life cycle is relatively long: polio viruses replicate in cell culture within six hours, whereas HCMV takes 72 hours to replicate.

Viral mechanisms to evade immune defence

The length of time it takes to replicate poses a particular problem when investigating the virus. HCMV replication is very complex, and interacts at many sites with cellular gene expression. The virus has developed effective mechanisms that enable it to evade the host’s immune defence, as it needs to prevent the infected cells from undergoing apoptosis before new functional viruses have been produced.

The photos show how HCMV-infected cells are removed from the cell assembly. © Mertens

Some of these evasion mechanisms are already relatively well understood. For example, the virus down-regulates MAC molecules on the cell surface that present virus fragments to the human immune system. In addition, the virus activates genes that prevent cellular apoptosis, as well as interfering with the formation of the extracellular matrix to prevent infected cells from anchoring stably in the tissue. In addition, the virus up- and downregulates VEGF receptors of the cell.

Association with other diseases?

HCMV has been detected in a renal artery organ model. The electron microscope image clearly shows the spiky spherical pathogen. © Mertens

The virologist from Ulm, who is also an influenza expert and a member of the Permanent Vaccine Commission at the Robert Koch Institute, has been concentrating mainly on three questions: He is interested to find out whether the complex interaction between virus and host also needs to be looked at in connection with other diseases, something that does not immediately spring to mind. His group of researchers found evidence that certain viruses were related in some way to the development of arteriosclerosis. Animal experiments showed that herpes viruses promoted the development of arteriosclerosis. Mertens assumes that infected cells or tissue turn into a kind of "proarteriogenic" state, but this is difficult to prove since there are no HCMV animal models available.

Virus uses cellular escort systems

Mertens’ second major research priority, dealing with the morphogenesis of HCMV, focuses on basic and applied research. Which viral proteins are needed by the virus and when are they needed for the virus to replicate “normally” in the host cell? An answer to this question would give insights into viral replication and the cellular mechanisms used by the virus. Mertens and his colleagues are principally focusing on cellular transport molecules, so-called escort systems. There is evidence that viruses such as HIV, herpes and HCMV use certain escort systems to complete their own replication.

Mertens is also interested in viral morphogenetics because he believes that this will provide him with new therapeutic targets, for example for the inhibition of viral replication. According to Mertens, the viral replication cycle needs to be divided into small partial processes in order to find targets of intervention.

Drugs with deficiencies

Currently used virustatic drugs prevent the replication of the viral genome and target the viral polymerase. These drugs are nucleoside analogues or pyrophosphate analogues. One drawback of such drugs is that they could provoke serious side effects and are not tolerated equally well by all patients.

But this is not the only thing that concerns Mertens. He has been able to show that HCMV becomes resistant to such drugs. The viral genes mutate spontaneously and become insensitive to the substances. He therefore sees it as extremely important to broaden the spectrum of anti-HCMV drugs and develop new substances that target different molecules and do not automatically lead to cross-resistance.

UL 97 – a new target for antiviral therapies?

When analysing the viral resistance mechanisms and their associated mutations, Mertens and his colleagues discovered a potential target for antiviral therapies, a viral enzyme called UL 97. UL 97, a kinase that has been known for 20 years, is responsible for the phosphorylation of proteins. Mertens and his team discovered that the viral UL 97 kinase “accidentally” phosphorylates certain nucleoside analogues used for the therapy of HCMV infections.

Phosophorylation makes a nucleoside analogue virus-selective. If the nucleoside analogue is brought into an infected cell, only this cell will produce the viral enzyme, which will then phosphorylate the nucleoside analogue into a mononophosphate. In order to be incorporated into the viral DNA by the polymerase, three phosphate groups must be added to this molecule

Database for researchers around the world

Mertens hopes that once the function of this kinase is understood in detail, the researchers will have the necessary information to be able to interfere with its function. At present, Mertens’ team is working with Hans Armin Kestler of the Institute of Neuroinformatics at the University of Ulm to set up a database containing all the potential mutations of this protein. Once it is completed, the database will be made available to researchers around the world. The database is designed to support researchers in assessing the sensitivity of the virus to UL 97.

If it transpires that UL 97 is required for viral replication, the researchers would then have a new therapeutic target, which, beside its ability to phosphorylate proteins, would have the advantage that it targets a viral mechanism that is different from that of the viral polymerase. But a lot of basic research is still required. The researchers will have to test protein kinase inhibitors for their antiviral effects. And of course, the target kinase inhibitors must not interfere with the protein kinases of the host cells.

No vaccine, no clinical candidates

In theory, all viral genes involved in the replication of the virus might be potential therapeutic targets. But finding this out requires time-consuming and expensive high-throughput molecule screening processes. Some pharmaceutical companies are conducting such screenings, some substances are already in the development pipeline and some are already being tested in clinical trials on humans. But due to the complexity of the virus, this is a very complex process. Attempts to develop a vaccine for HCMV have been unsuccessful, and there is no vaccine currently being tested in advanced clinical trials.

Differentiation stage of the cell might help the viruses to hide

Little is still known about the molecular switches that cause the virus to go into a dormant state before being subsequently reactivated. The researchers assume that the differentiation state of certain cells plays a role in this. Whilst monocytes are able to take up viruses, they prevent the viruses from replicating. On the other hand, differentiated macrophages are able to support the viral replication cycles. Mertens concludes from such laboratory findings that a combination of viral properties and cellular differentiation stages (including consequences such as cytokine release or alloreactions) is required for the creation of an environment that enables certain molecular switches to be turned on or off. Mertens is also sure that the identification of such switches requires intensive cooperation between virologists and cell biologists.

Selected literature:
Chevillotte M, Schubert A, Mertens T, von Einem J.: A fluorescence-based assay for phenotypic characterisation of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness, in: Antimicrob Agents Chemother. 2009 Jun 22.

Schreiber A, Härter G, Schubert A, Bunjes D, Mertens T, Michel D.: Antiviral treatment of cytomegalovirus infection and resistant strains, in: Expert Opin Pharmacother. 2009 Feb;10(2):191-209.

Chevillotte M, Landwehr S, Linta L, Frascaroli G, Lüske A, Buser C, Mertens T, von Einem J.: Major tegument protein pp65 of human cytomegalovirus is required for the incorporation of pUL69 and pUL97 into the virus particle and for viral growth in macrophages, in: J Virol. 2009 Mar; 83(6):2480-90.

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