A plasmid on the road to becoming a virus

Origin of virusesPlasmids have been discovered that can move from cell to cell within membrane vesicles in a species of Archaea (link to paper). They provide clues about the origin of virus particles.

Electron microscope analysis of the culture medium from Halobrum lacusprofundi R1S1, an Archaeal strain from Antarctica, revealed spherical particles which were subsequently shown to contain a 50,000 base pair circular double-stranded DNA molecule. When added to H. lacusprofundi, the purified membrane vesicles entered the cells and the DNA replicated.

Nucleotide sequence analysis of the plasmid within the membrane vesicles revealed 48 potential protein coding regions and an origin of DNA replication. None of these proteins showed any similarity to viral stuctural proteins, leading the authors to conclude that these particles are not viruses.

Many of the proteins encoded in the plasmid DNA were found in the membrane vesicles. Some of these are similar to cell proteins known to be involved in the generation of membrane vesicles. However no DNA polymerase-like proteins are encoded in the plasmid. These data suggest that the plasmid encodes proteins that generate, from the membranes of the cell, the vesicles needed for their transport to other cells. However, replication of the plasmid is carried out by cellular DNA polymerases.

It is likely that the plasmid-containing membrane vesicles are precursors of what we know today as virus particles. It is thought that viruses originated from selfish genetic elements such as plasmids and transposons when these nucleic acids acquired structural proteins (pictured; image credit). Phylogenetic analyses of the structural proteins of many enveloped and naked viruses reveal that they likely originated from cell proteins on multiple occasions (link to paper).

The membrane-encased Archaeal plasmid seems well on its way to becoming a virus, pending acquisition of viral structural proteins. Such an early precursor of virus particles has never been seen before, emphasizing that science should not be conducted only under the streetlight.

TWiV 437: Kathy’s new spindle virus

The TWiVsters reveal new giant viruses that argue against a fourth domain of life, and discovery of viruses in the oceanic basement.

You can find TWiV #437 at microbe.tv/twiv, or listen below.

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TWiV 367: Two sides to a Coyne

On episode #367 of the science show This Week in Virology, two Coynes join the TWiV overlords to explain their three-dimensional cell culture model of polarized intestinal for studying enterovirus infection.

You can find TWiV #367 at www.microbe.tv/twiv.

Exaptation: A cell enzyme becomes a viral capsid protein

Alphalipothrixvirus virionThe acquisition of a capsid is thought to be a key event in the evolution of viruses from the self-replicating genetic elements that existed during the pre-cellular stage on Earth. The origin of viral capsids has been obscure because their components are not similar to cellular proteins. The discovery that a viral capsid protein evolved from a CRISPR-associated nuclease provides insight into how viruses emerged.

Thermoproteus tenax virus 1 (TTV1) infects the hyperthemophilic archaeon Thermoproteus tenax, which grows at 86°C. The enveloped virus particles are flexible filaments 400 nm long and 40 nm in diameter (illustrated; image credit) built with four capsid proteins, TP1-TP4. The basic proteins TP1 and TP2 bind the 16 kb double-stranded DNA genome to form the nucleocapsid.

Thirty years after the discovery of TTV1, the capsid proteins remained ORFans – meaning that they had no sequence homology with viral or cellular proteins. Recently a more sensitive homology analysis revealed that TP1 is similar to Cas4, a nuclease that is a part of the prokaryotic CRISPR-Cas defense system.

Although TP1 clearly matches the Cas4 protein, it is not complete: codons at the carboxy-terminus are missing. A re-examination of the TTV1 genome sequence revealed a previously undetected open reading frame of 74 codons just downstream of the TP1 gene which are the missing C-terminal residues of the Cas4 nuclease. It is not known if this protein, called gp7, is produced in infected cells; it is not part of the virus particle.

Together the TP1 and gp7 proteins represent a full length Cas4 nuclease. TP1 is probably not catalytically active due to amino acid changes in the active site of the enzyme.

Why does TP1 lack the carboxy-terminal residues of Cas4? The amino terminus of the TP1 protein comprises a positively charged surface that might be involved in binding the viral DNA genome. The same surface in Cas4 is covered by the carboxy-terminal domain of the protein. This observation suggests that transformation of Cas4 from a nuclease into a viral capsid protein probably required removal of this shielding domain, so that the protein could bind the DNA genome.

How did a nuclease become a viral capsid protein? An ancestor of TTV1 might have encoded a Cas4-like protein with nuclease activity with a role in genome replication or repair. Mutations causing loss of nuclease activity might have been followed by truncation of the protein to expose the DNA binding domain, which then became a viral capsid protein. Support for this idea comes from the observation that a Cas4-like protein encoded in the genome of another archaeal virus, the rudivirus SIRV2, has nuclease activity.

Exaptation, a change in the function of a protein during evolution, is known to have taken place in the viral world. The case of Cas4 and TP1 shows that capsid components can evolve from proteins with a very different function.

TWiV 342: Public epitope #1

On episode #342 of the science show This Week in Virology, the TWiVniks discuss the structure of a virus that reproduces in an extreme environment, long-term consequences of Ebolavirus infection, and VirScan, a method to identify the different virus infections you have had in your lifetime.

You can find TWiV #342 at www.microbe.tv/twiv.

Viruses in the extreme

RudivirusMany microbes live in extreme environments, encountering conditions that are very hot, very cold, highly acidic, or very salty. The viruses that infect such microbes must also be able to retain infectivity in extreme conditions. How do they do it?

Clues come from the observations that the genomes of viruses that infect Archaea in extreme geothermal environments encode proteins that have never been seen before. The idea is that such unusual proteins must endow these viruses with the ability to maintain infectivity under extreme conditions.

The hosts of Rudiviruses (rudi=small rod in Latin), the Archea Sulfobolus islandicus, live at high temperatures (80° C) and low pH (3.0). These non-enveloped viruses consist of double-stranded DNA wrapped in a helical manner with thousands of copies of a 134 amino acid protein (illustrated; image credit). The three-dimensional structure of Sulfobolus islandicus rod-shaped virus 2 (SIRV2) reveals a new type of organization of virus particles, and provides clues about how it retains infectivity in extreme environments.

Resolution of the SIRV2 structure reveals that it consists of dimers of a single protein which forms helices that are tightly wrapped around the DNA genome. The result is a coiled DNA protected by a coat of protein that stabilizes and protects the genome. Without DNA, over half of the capsid protein is unstructured. Only in the presence of DNA does the viral protein form an alpha helix that wraps around the nucleic acid.

The DNA genome of SIRV2 is in the A-form, in contrast to B-form DNA which is found in most other organisms. The two types of DNA differ in their geometry and dimensions. It was previously thought that A-DNA occurs only when the nucleic acid is dehydrated.

These two usual properties of SIRV2 are also found in gram positive bacteria which form desiccation and heat resistant spores when starved of nutrients. Sporulation is accompanied by a change in the bacterial genome from B-DNA to A-DNA, which is caused by the binding of small acid-soluble proteins. Like the SIRV2 capsid protein, small acid-soluble proteins of spore-forming bacteria are unstructured in solution, and become alpha helices when bound to DNA. These observations suggest that binding of the SIRV2 capsid protein changes the viral DNA to the A-form, conferring stability in extreme environments.

An RNA virus that infects Archaea?

Nymph Lake, Yellowstone National ParkEvery different life form on earth can probably be infected with at least one type of virus, if not many more. Most of these viruses have not yet been discovered: just over 2,000 viral species are recognized. While the majority of the known viruses infect bacteria and eukaryotes, there are only about 50 known viruses of the Archaea, and these all have DNA genomes. The first archaeal RNA viruses might have been recently discovered in a hot, acidic spring in Yellowstone National Park.

Archaea are single-cell organisms that are similar in size and shape to bacteria, but are evolutionarily and biochemically quite distinct. They inhabit a broad range of environments including those with extreme conditions such as high temperature, acidity, and salinity. Identification of archaeal RNA viruses is important because their study could provide information about the ancestors of RNA viruses that infect eukaryotes. Direct sequencing of viral communities from the environment, known as viral metagenomics, is one approach being taken to discover archaeal viruses.

The acidic (pH <4) and hot (>80°C) springs in Yellowstone National Park were examined for the presence of archaeal RNA viruses because these bodies of water contain mainly Archaea. Samples were obtained from 28 different sites and extracted nucleic acids were treated with DNAase (to remove DNA genomes) and then reverse transcriptase (to copy RNA to DNA). If reverse transcription was reduced by treatment with RNAse, it was concluded that the sample contained mostly RNA. The results narrowed the sample size to three, all from Nymph Lake. New samples obtained twelve months later also showed a predominance of RNA and were used for metagenomic analysis by deep sequencing.

Analysis of the RNA viral sequences revealed coding regions for a predicted RNA dependent RNA polymerase (RdRp), a hallmark of RNA viruses. One assembled sequence of 5,662 nucleotides, believed to be a complete viral genome, encodes a single open reading frame containing a RdRp and a putative capsid protein similar to that of the positive-strand RNA containing nodaviruses, tetraviruses, and birnaviruses. Another viral sequence encoded a protein with 70% amino acid homology to the predicted RdRp. The sequences are from a novel virus which does not belong to any known virus family.

These results clearly show that at least two related but distinct RNA viruses are present in Nymph Lake. However whether or not the hosts of these viruses are Archaea or Bacteria cannot be determined by these metagenomic analyses. What is needed to resolve this question is old-fashioned virology:  isolating RNA virus particles that can infect an archaeal host and produce new infectious viruses.

B Bolduc, DP Shaughnessy, YI Wolf, EV Koonin, FF Roberto and M Young J. Virol. 2012, 86(10):5562. DOI: 10.1128/JVI.07196-11.

TWiV 195: They did it in the hot tub

On episode #195 of the science show This Week in Virology, the complete TWiV team meets with Ken Stedman to discuss the discovery in Boiling Spring Lake of a DNA virus with the capsid of an RNA virus.

You can find TWiV #195 at www.microbe.tv/twiv.

Prokaryotes considered

prokaryoteAs a college biology major during the 1970s I was taught that cells in which the genetic material is separated from the cytoplasm by a nuclear membrane – such as those of animals, fungi, plants, and protists – are called eukaryotes. In contrast, the DNA of bacteria is not bounded by such a structure, and hence these microbes are called prokaryotes, a name that means ‘before the nucleus’. This concept was accepted by biologists until the late-1970s, when Carl Woese used ribosomal RNA sequences to deduce the relationships among living organisms. He found that microorganisms previously thought to be bacteria, because they have no nucleus, were no more related to bacteria than to eukaryotes. He proposed that living organisms should be classified into three lineages, now called bacteria, archaea, and eukarya. Nevertheless, the prokaryotic classification is still used by many biologists. The following letter from Elio Schaecter, sent to TWiV, explains why:

“Regarding your discussion of the term prokaryote in TWiV #93, I want to pipe in as a combatant in the “P word” wars. I am firmly in the camp of the users of the term. Although the term has carried a phylogenetic burden, meaning that it originally implied a close evolutionary relationship between the Bacteria and the Archaea, no one I know uses it in that sense now. Among biologists, the three domains model is widely accepted, in fact, not even discussed. It’s true that there are leftover people who think that the prokaryote/eukaryote divide denotes a single evolutionary cleft, but that’s simply because any concept of science takes time to filter out.

“I maintain that the term, used in a broad sense, is extraordinarily useful. In a college textbook I co-authored called “Microbe”, we used the P word some 300 times. How come? Had we not used it, we would have had to say “Bacteria and Archaea” that many times (and being parsimonious of verbiage, we eschewed that). This usage illustrates the reality that these two groups of microbes, though they likely diverged very early on in evolution, share a large number of common properties. Their sizes tend to overlap, their overall body plan is generally very similar, they often occupy the same habitats, they share many homologous genes. Presented with an EM thin section, you could not tell a typical bacterium from a typical archaeon. So, dissimilar as they may be in one sense, they are very similar in a number of important attributes. Saying “prokaryotes” is much like saying “animals” or “plants,” large groups that are extremely heterogeneous and that diverged a long time ago (although certainly not as far back as the prokaryotes and eukaryotes). I agree, there is danger in the P word being misunderstood out in the big wide world, but there is none within the family of biologists.

“Anyhow, the battle has been met and, yeah!, the victors are clearly the users of the word prokaryote. The term is found all over the place, notwithstanding the astonishing campaign waged against it. Just look at titles of recent articles in major journals.

“There is a more serious issue. Making phylogeny the overarching master of relatedness is readily justified if one thinks in these terms only. But isn’t ecology just as important to understand biological behavior and relatedness? There is a tyranny to phylogeny, which demands that you view the world of living things in terms of where they came from, not what they are doing now.”

What does this nonmenclature issue have to do with virology? According to Patrick Forterre:

The discovery of unique viruses infecting archaea also corroborates the three domains concept from the virus perspective. Indeed, most viruses infecting archaea have nothing in common with those infecting bacteria, although they are still considered as “bacteriophages” by many virologists…David Prangishvili and myself have thus suggested to classify viruses into three categories, archaeoviruses, bacterioviruses and eukaryoviruses.

Woese, C. (1977). Phylogenetic Structure of the Prokaryotic Domain: The Primary Kingdoms Proceedings of the National Academy of Sciences, 74 (11), 5088-5090 DOI: 10.1073/pnas.74.11.5088

Prangishvili, D., Forterre, P., & Garrett, R. (2006). Viruses of the Archaea: a unifying view Nature Reviews Microbiology, 4 (11), 837-848 DOI: 10.1038/nrmicro1527

A new type of enveloped virus?

All known virus particles can be placed into one of two general categories: enveloped or non-enveloped. Viruses that fall into the former category are characterized by a lipid membrane derived from the host cell, and one or more nuclecapsid proteins that interact with the viral genome. A virus that infects an archaeal host may constitute a new category of enveloped viruses. It comprises a membrane vesicle that encloses a circular ssDNA genome which is devoid of nucleic acid-binding nucleoproteins.

Examples of enveloped virions that contain nucleoproteins are shown in the figure below. These include influenza virus (left), a simple retrovirus (center), and a togavirus (right).

The influenza virion contains segments of viral RNA bound to four different proteins. Retroviral RNA is bound to a nucleocapsid protein which in turn is enclosed in a capsid, while togavirus RNA is located within an icosahedral shell.

Until recently, it was believed that the genome of all other known enveloped DNA and RNA viruses is always associated with one or more viral proteins. This belief may be changed by the isolation, from a solar saltern in Trapani, Italy, of a virus that infects the archaeal species Halorubrum. Salterns are multi-pond systems in which sea water is evaporated to produce salt. In such hypersaline envrionments, Archaea predominate, and about 20 archaeal viruses have been isolated from these locations.

The virus isolated from the Italian saltern is called Halorubrum pleomorphic virus-1, or HRPV-1. Biochemical analyses of the virion show that it is composed of lipids and two structural proteins, VP3 and VP5. The genome is a circular ssDNA about 7 kb in length with nine open reading frames. The virion architecture is unique: it is composed of a flexible membrane (hence the designation pleomorphic) that contains external spikes of the VP4 protein, and is lined on the interior with VP3. The viral DNA is apparently not bound to any proteins in the virions.

At the upper left is my depiction of the appearance of HRPV-1. The diagram was produced by deleting the internal proteins and nucleic acid of a simple retrovirus and replacing these with a ssDNA genome. The HRPV-1 VP4 spikes and the internal VP3 proteins are present, but no proteins are bound to the viral genome. Whether or not the VP4 spikes are oligomeric as shown is unknown.

Most enveloped viruses acquire their lipid membrane by budding from the host cell, and a similar mechanism could account for the formation of HRPV-1 virions. In the absence of a nucleoprotein, it is not clear how the viral genome would be specifically incorporated into the budding envelope. Another condundrum is how the virions would pass through the proteinaceous layer that covers the archaeal host cell.

Whether HRPV-1 is representative of a new kind of virus lacking nucleocapsid protein will be revealed by the study of other pleomorphic enveloped viruses. Candidates include bacterial viruses that infect mycoplasmas, and another pleomorphic haloarchaeal virus isolated from a different Italian saltern, Haloarcula hispanica pleomorphic virus 1.

Pietila, M., Laurinavicius, S., Sund, J., Roine, E., & Bamford, D. (2009). The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes Journal of Virology, 84 (2), 788-798 DOI: 10.1128/JVI.01347-09