Viruses More Friends than Foes

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Review DOI: 10.1002/elan.201900604

Viruses More Friends than Foes Karin Moelling*[a]

Abstract: Viruses are normally defined as pathogens and have a bad reputation because of pandemics such as Influenza, HIV/AIDS, Ebola, and SARS. Most viruses are, however, not enemies or killers but play important roles in the origin, development and maintenance of life of all species on our planet. This is new information we learnt by new technologies such as sequencing. Viruses are the most successful species on Earth, they are ubiquitous, in the oceans, in our environment, in animals, plants, bacteria, up in the air, perhaps even in the universe, within our body and even as part of our genomes. They influence our health, our well-being, mental properties, our gut microbiota including obesity,

and may help to cope with multi-drug-resistant bacteria. There the phages, viruses of bacteria, raise hopes. Viruses built our immunity: viruses protect against viruses. We do not have to lay eggs – thanks to viruses! They are the drivers of evolution and adaptation to environmental changes, also e. g. in plankton. The success story of viruses started about 3.5 billion years ago when life began. Newly discovered giant viruses are almost bacteria in their composition, suggesting that the borderline between dead matter and life is continuous. There are many open questions – how did life begin, is there life on exoplanets, how to find it? Are virus-like elements, viroids, important for the origin of life? Will viruses eliminate mankind [1]?

Keywords: Viruses · microbiome · viral sequences in all species · origin of life · not causing diseases

1 Viruses on our Planet There are about 1032 viruses on our planet, making them the most successful species. Most of them do not cause diseases. Their reputation as pathogens is based on the history of medicine. Yet, only about 1000 viruses are known to cause diseases in humans, some of their pathogenicity is due to environmental effects. The phrase “I got a cold” is indicative for an altered, a cold environment, which can promote a viral disease by activating rhinoviruses or Influenza viruses. Also lifestyle of the host, of people, can play a role as in the case of HIV [1]. Viruses have been disastrous during the history of mankind – such as Influenza and HIV, which killed millions of people. Viruses can spread around the world within hours with the increased mobility by airplanes. Indeed, newly emerging viruses, such as West Nile Fever Virus, conquered the United states in a few years. Viruses are present in every single species on our planet, normally without notice. They populate the soil, the oceans, the air, our human body and even our genome. Viruses interact with their host and are able to transfer genetic material across various species. Thus the genetic composition of viruses changes and is a combination derived from many hosts – raising the problem, of how to define or type a virus on the basis of its genetic material. The mechanism of horizontal gene transfer is the basis for the complexity of all genomes, including the human genome. Humans evolved late during the history of life on our planet, the microbes and fungi were present for billions of years before us. Homo sapiens entered the scene around 300.000 years ago and had to cope with all the microbes. www.electroanalysis.wiley-vch.de

Thus, all mammals and us had to adjust and learn how to coexist with existing microbes. Our genome reflects all the encounters. When it was sequenced, it was indeed one of the biggest surprises that the it consists of almost 50 % of retroviruses or retrovirus-like sequences. Sequencing of whole genomes was first applied to the human genome. It revealed, that the human genome is composed of up to 80 % of viral information [1]. One may ask, whether this was 100 % earlier during evolution – and whether viruses are our oldest ancestors [1]?

2 Endogenous Viruses in our Genes The presence of viruses inside genomes raises the question about their biological role – which is certainly not primarily to cause diseases. Instead, viruses are drivers of evolution. The majority of viruses inside mammalian genomes are the retroviruses, which contain RNA as primary genomes but adopt as intermediate a DNA provirus of about 10 viral genes during replication. This DNA provirus can then integrate into the human [a] K. Moelling Inst for Medical Microbiology, University Zürich, Gloriastr 30, 8006 Zürich, Switzerland and Max Planck Institute for Molecular Genetics, Ihnestr 73, 14195 Berlin, Germany E-mail: [email protected] © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Review genome as additional inheritable genetic information. RNA is a versatile molecule which can change, while a DNA copy is more stable and conserves the genetic information. Integrated viruses are so-called “endogenous viruses”. They can interact with the existing cellular genes in their neighbourhood in several ways, the integration can do some harm by disrupting preexisting genes, or the viral regulators (promoters) can deregulate cellular genes besides regulating their own genes. This may cause cancer or lead to the opposite, a genius, a Mozart or Einstein [1]! We analyzed a mouse-like human endogenous retrovirus HERV-K, which can turn off a tumor suppressor gene and thereby induce malignant transformation of a cell. However, this appears to be a very rare event [2]. Endogenous viruses are present with almost millions of copies in our genomes [3]. The endogenous DNA proviral genomes accumulate mutations over time, if they are not needed for some functions and not selected for stability. Indeed, some mutated proviral sequences are 45 Mio years old. They were analyzed by “repair” in the laboratory and such reconstructed viruses gave rise to a real infectious virus, named Phoenix [4]. This proved that the viral sequences in mammalian genomes entered by real infections. It should be stressed that this is an extremely rare event during the life span of a human or other mammals. The virus normally does not enter germ cells but somatic cells. Yet, this rare event is the basis for the presence of retroviral sequences in mammalian genomes. An endogenous virus protects against foreign viruses. This antiviral defense is what we define as immunity. A virus excludes other viruses and thereby generates immunity in the infected cell. A virus protects against a virus [5]! Another very surprising effect was caused by a retroviral integration into the germline of mammals: The virus protects the embryo against the immune defense of the mother and therefore mammals do not have to lay eggs and mothers can hatch embryos inside of their body [6]. The immune suppressed mother can do that in her womb now. The protein which causes the mother‘s immune suppression and allows the embryo to grow, is almost identical to one of the HIV proteins involved as cause of AIDS. Yet this happened 65 Mio years ago. This proves that viruses can influence evolution [1, 7]. The infection of germline cells is a major concern for gene therapy in medicine where often viruses serve as transfer vehicles for therapeutic genes. The germ line is by the self-restriction of scientists a “no-no”– yet with the CRISPR/Cas – treatment of germ cells in China, this tabu was broken. We do not want to alter genes of the next generation, but the Chinese did not sign this agreement [1].

3 Transposable Elements Not only exogenous viruses interact with cellular genomes but the cellular DNA can also undergo modifications. A double-helix cannot easily allow mutations by single www.electroanalysis.wiley-vch.de

nucleotide changes, because of the second stabilizing strand. This is in contrast to single-stranded RNA, which is a characteristic of many highly variable and therefore dangerous viruses, because they escape immunity. Different mechanisms exist for modifying DNA. They resemble those applied by the “word program” on a laptop computer, which indeed resembles word processing applied to genetic letters, the sequence. Processing can occur by “cut-and-paste” or “copy-and-paste”, corresponding to displacement of genetic fragments or their duplication. In both cases the DNA fragments are reinserted into the genomes, the former text. These genetic actions are designated as “jumping genes” or more precise as transposable elements (or transposons) or retrotransposons, respectively. Retrotransposons resemble a retroviral replication including integration. It involves a step mediated by the “Reverse Transcriptase”, the enzyme which reverse transcribes RNA into DNA. The only missing element in this mechanism is a protein coat, which makes viruses mobile and allows them to leave a cell and enter a new one. Transposable elements are thus almost “prisoners”, locked inside a cell without the possibility to leave it. A superinfecting virus can overcome this by sharing its coat proteins. Protection of a cell by a virus from superinfection by another virus is designated as superinfection exclusion, which means “no entry”! This can be followed today with koalas, where a fortuitously endogenized monkey virus made the koalas resistant against infection by other viruses in a rather short period of time, in about 100 years [5]. We also know about monkeys which can be infected by a monkey-virus, the simian-specific HIV, called SIV, without causing a lethal disease. Many monkeys may have died unnoticed. The mechanism evolved is superinfection exclusion by the first virus against later ones. May be, one day humans become resistant against HIV – as has happened during evolution with many viruses in our genome and in the monkeys against SIV [5]. Viruses are a very potent driver of evolution, because many genes enter the genome with a single strike. This is not what Darwin could have envisaged when he described the Eevolution of species: he observed many minor evolutionary changes but no big ones, no jumps [8]!

4 Giant Viruses Viruses are normally thought of as small, smaller than bacteria and unable to replicate autonomously, but dependent on a host protein synthesis machinery. Yet, viruses are not necessarily small. Since a decade ago a new type of viruses has been identified, the giant viruses. Their genomes can be huge and the viruses exceed in size many of the known viruses and even some bacteria. Furthermore, these giant viruses contain almost a full set of genes and the tool kits required for protein synthesis – which was very surprising initially [9]. Thus the transition from viruses to bacteria is continuous. This also means that the transition from dead matter to living matter is

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Review continuous, not simply two separate categories, not simply small and large! There is no sharp border between viruses and bacteria – even though only bacteria are considered alive. The definition is mainly based on the ability of producing their proteins themselves as autonomous entities. However, there is no autonomous life on the Earth which can get away without help or support or food from outside. Independent growth as criterion of life was therefore rejected by Steve Hawking [10]. Furthermore, the giant viruses are “almost” bacteria in that they have many building blocks – but not all of them – required for protein synthesis [11]. One may ask whether they where blocked on their way to become bacteria and remained unfinished in the course of evolution or whether they regressed from bacteria to simper forms of life? Gains of genes is a well-known phenomenon, yet the loss of genes is often forgotten about, but is a frequent consequence of luxurious environments. The life-style of an organism is determined by its surroundings: If one puts a microorganism into a paradise-like environment, it can give up non-essential genes. This reduced entity was no longer coding for genes and rapidly replicating, designated as Speigelman‘s Monster [12, 13]. A good example are the mitochondria. They have evolved from a former bacterium, which became a power house in our cells. For that it gave up almost 3000 genes and kept 300 special ones to fulfil a single important function, to generate energy for its host. Many other functions were supplied by the host or delegated to him [14]. This makes me speculate that viruses may have been independently replicating entities initially, which grew up to cell-like structures. Then, some viruses changed their life-style and became parasites, as we know them today. They have been described as metabolically relevant entities, which can dissolve their host cells such as algae, and thereby help recycling of nutrients in the oceans. The giant viruses are activated by stress – a reaction which resembles stress in bigger animals: no food, too many nutrients, no space, wrong temperatures – this can activate the viruses. They can lyse chalk algae which ended up after millions of years as the white cliff of Dover or the white rocks on the island of Rugen. Giant viruses infect amoebae, which are very big themselves and where extensive gene exchange can take place, leading to the giant genomes of the giant viruses. No diseases are known which can be attributed to them [1, 9].

5 Phages, the Viruses of Bacteria The largest group of viruses are the viruses of bacteria which have a special name, phages or bacteriophages. They are most abundant everywhere on our planet, in the oceans, the soil, the air and inside and outside of our body. Most of them are inside bacteria, their natural host. They were discovered more than 100 years ago by the Canadian researcher Felix d'Herelle at the Institute Pasteur in Paris. He saw that a lawn of bacteria in a petri dish was dissolved by phages – which he isolated by www.electroanalysis.wiley-vch.de

filtration from the feces of a soldier who suffered from dysentery. He used the phages as a drink to cure himself. We isolated some phages from human feces of a patient, who was sick with multidrug resistant bacteria present in her feces and received a fecal transfer from a healthy donor [15]. The phages behave like viruses, they can coexist with their host, without being noticeable. But upon stress they can replicate and lyse the bacterial host. 80 % of the bacteria in the oceans are lysed per day. This could and should be exploited to kill resistant bacteria. The history of phages dates back thousands of years. A curing activity had been detected in the Ganges River as the basis for the rituals of Hindus, who drink and submerge in the river water, which proved to be indeed active against bacteria. This activity was shown by a British biologist Hankin to be thermo-sensitive which disappeared upon heating [16]. The effect was due to phages which were only discovered 20 years later by d'Herelle. He traveled wherever some infectious disease occurred and treated the patients with phages, which he isolated on the sites, in South America, Africa (Ruanda, Burundi, Congo), Mexico and passengers on a French ship passing through the Suez Canal. He was invited to Yale University in the USA and Canada and then to Georgia. Together with Georgi Eliava they established phage therapy in Tsiblisi at the Eliava Institute in 1936, which still exits until this very day. Up to 1200 employees were producing tons of phages in peak times. They produced pills, band aids, and treated 18.000 soldiers in the Finnish-Russian War in 1939 with a phage cocktail against anthrax with 80 % success rate, which means avoiding amputations. In 1963 as many as 30.000 children were treated some even with phage as a prophylactic, which required high doses of phages, frequent application and did not turn out useful unless during ongoing epidemics [17, 18]. Antibiotics were not easily available in The Eastern World, which kept phage therapy going as an option. Much of this seems forgotten about. Now phages have a come-back during times of multidrug-resistant bacteria in hospitals with people dying. Three recent cases were spectacular enough to raise attention. Phages were selected to fit to the bacterial infections and were even injected into the patients which saved their lives within a few days [19]. One can go fishing in the sewage of hospitals – as in the Ganges River – and isolate the phages, which are present in lage quantities wherever the repective bacteria occur. The phages can then be processed, typed by sequencing, and grown up for further use. The regulatory agencies, who have to approve novel therapies, slowly notice the relevance of this procedure, which cannot easily be approved by the present drug regulations. Phage therapy requires different approvals – and perhaps a new definition other than a drug [19]! It is not trivial to isolate and characterize phages from patients. We isolated phages from a patient who had received a fecal transfer because he suffered from an untreatable infection with Chlostridium diffice.

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Review The phages occur in small amounts and are difficult to detect and require amplification [20].

6 Are Viruses Alive? Can they be the Origin of Life? Will they Kill Mankind? Viruses were defined as a composition of genetic material and some kind of protection by proteins or lipids or membranes taken from the host cell during budding. The structure resembles that of an apple: seeds with genetic material are surrounded by the apple and a coat. Textbooks describe viruses as parasites which depend on energy supply from a host: However – there is no living entity on our planet which can get away without energy supply from the outside. The question then is, whether viruses are alive? There are dozens of definitions about life. NASA suggested: a self-replicating entity able to evolve. Then viruses are alive – this was proposed by S. Hawking [10]. Since viruses are the smallest entities on Earth which can replicate and evolve, which means adjustment to the environments, the question arises, whether they could have been the origin of life. Ribozymes, RNA enzymes, have been discussed as the first replicating units, and were also designated as viroids, virus-like entities [21, 22, 23, 24]. (Figure 1). The question on the origin of life has been raised repeatedly and now becomes a focus of interest

because of the exploration of the Moon and Mars. The European Space Agency, ESA, attached about 20 biological specimens for experiments onto or into the International Space Station, ISS [25]. They tested whether life can “survive” there in 400 km altitude being exposed to extreme temperatures and genotoxic radiation. Furthermore, exoplanets have been discovered about 20 years ago and their number is estimated to about 1022 [26]. These exoplanets orbit around their own suns, like the Earth around our sun. The number is so high, that the probability to allow live to evolve or exist there, is high. There are even theories since decades which suggest that life on Earth originates from the universe outside of the Earth, the pansperm theory. There are arguments against it because radiation from space over long distances would not allow traveling of living entities like life on earth. The pansperm theory still has many followers today [27, 28]. There are meteorites which contain up to 90 amino acids, even though we have only 20 as the basis of life on Earth. Then meteorites contain more than 13.000 carbon-based compounds and building blocks for nucleic acids [29]. Life on other planets could be different than life here on our Earth. The smallest replicating and mutating elements on Earth are viroids, virus-like structure. They are also designated as ribozymes, an abbreviation of RNA and enzyme – because these are not protein-based enzymes but consist of catalytically active RNA. From a large mixture of random RNAs (1014, 80mers), viroids/

Fig. 1. Model according to [24] with viroid/ribozme and tRNAs from a quasispecies as essential elements for protein synthesis from a virus to an early cell. Protein synthesis depends on the ribozymes and tRNAs, both non-coding RNAs, which are essential parts of our world till today. Viruses became internalized as aparasites and developed to the fastest replicating and most abundant species on Earth. www.electroanalysis.wiley-vch.de

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Review ribozymes or tRNAs can evolve [30, 31, 32]. They are noncoding RNA, that means they do not contain the nucleotide triplets which correspond to amino acids. They may be something like analphabets! The viroids are made of closed circular hairpin-loop structures. Protein synthesis in our cells depend on the action of ribozymes, the most essential constitutent of ribosomes till today (Figure 1). Viroids/ribozymes may be model structures of early life-like elments, something very close to the origin of life. However, on other planets they may look very different in terms of their chemical composition, but may be similar in structure and function such as self-replicating [6, 33]. They can grow and increase their information content and may have become precursors of cells. However, there is a hen – and – egg problem: if the viroids were first and autonomous, how come that the viruses today are parasites and depend on the metabolism and energy supply of a host cell – if they generated the first kind of pre-cells themselves? This may be explained by the two genetic driving forces, increase of genetic material and decrease. Depending on the surrounding, gain and loss of genes can occur. Sparse living conditions lead to increase of genetic complexity. Affluence in contrast, can cause loss of genes and lead to dependence. Intracellular life is like paradise for a virus but makes it a parasite [8, 12, 13]! Will viruses destroy mankind one day? This question is often raised. Viruses quickly adjust to their environment. When they have killed most hosts and they cannot find a host for replication any more, they will adjust their life-style by homing and adjustment to their host. as a means of survival. Thus, viruses will most likely not lead to the extinction of their hosts, such as people. They will turn into friends. What will happen in another 4 Billion years by the blow-up of the sun as a dying star getting close to the Earth. We all will be burnt.

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© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA

Received: October 6, 2019 Accepted: October 20, 2019 Published online on November 26, 2019

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