A viral nucleus

Cell typesA unique feature of eukaryotic cells, which distinguishes them from bacteria, is the presence of a membrane-bound nucleus that contains the chromosomal DNA (illustrated; image credit). Surprisingly, a nucleus-like structure that forms during viral infection of bacteria is the site of viral DNA replication (link to paper).

During infection of Pseudomonas bacteria with the phage 2O1phi2-1, a separate compartment forms in which viral DNA replication takes place. A phage protein, gp105, makes up the outer layer of this compartment, which initially forms near one end of the cell, and then migrates to the center. The migration of the compartment takes place on a spindle made up of the tubulin-like protein PhuZ.

In addition to viral DNA, certain proteins gain entry into this compartment, including viral proteins involved in DNA and mRNA synthesis, and at least one host cell protein. Other proteins, such as those involved in translation and nucleotide synthesis, are excluded. This compartmentalization very much resembles that of the nucleus of eukaryotic cells.

Packaging of the viral DNA takes place on the surface of the viral nucleus. Empty phage capsids form at the bacterial cytoplasmic membrane, then migrate to the compartment where they attach firmly to the surface. By an unknown mechanism, DNA moves from the compartment into the capsid. Then  capsids are released from the surface to further mature in the cytoplasm. The completed phages are released from the cell upon bacterial lysis.

These fascinating observations raise a number of unanswered questions. Does infection with other phages lead to assembly of a viral nucleus? How do molecules selectively move in and out of the structure?

Perhaps the most interesting question relates to the origin of viruses and cells. According to one hypothesis, self-replicating, virus-like nucleic acids might have first appeared on Earth, followed by cells without a nucleus. Was the nucleus a viral invention?

Top secret, viruses with RNA genomes!

Top secret!Today it is well known that viruses may contain DNA (poxvirus, mimivirus) or RNA (influenza virus, Zika virus), but for many years it was thought that genomes were only made of DNA. The surprise at finding only RNA in a virus is plainly evident in a 1953 letter from Harriett Ephrussi-Taylor to James D. Watson (pictured, Cold Spring Harbor Archives Repository*).

While DNA was discovered in the late 1800s, its role as genetic material was not proven until the famous experiments of McLeod, Avery, and McCarty, published in 1944. They showed that DNA from a strain of Pnemococcus bacteria that formed smooth colonies, when added to a rough colony former, produced smooth colonies.

By this time many viruses had been identified, and it was assumed that their genetic information was DNA. The ‘kitchen blender’ experiments of Hershey and Chase in 1952 proved that the genetic information of bacteriophage T2 is DNA. Watson and Crick proposed the double-helical structure of DNA in 1953, and a few years later published the Central Dogma, which suggested that information flowed in biological systems from DNA to RNA to protein.

Amidst all these experimental findings, which gave rise to the field of molecular biology,  comes the note in 1953 from Ephrussi-Taylor to Watson. Under the heading TOP SECRET she writes:

Burnet swears, from work in his lab, that flu virus has principally, if not exclusively RNA. Suspects the same for polioviruses. ??

During her career, Dr. Ephrussi-Taylor carried out work on bacterial transformation by DNA and was knowledgeable about its history as genetic material. Frank Macfarlane Burnet was an Australian immunologist who worked on influenza virus early in his career.

By the 1950s many viruses had been isolated which we now know have genomes of DNA (bacteriophage, poxvirus) or RNA (yellow fever virus, poliovirus, influenza virus). But it was the first virus discovered – tobacco mosaic virus, in the 1890s – that lead the way to establishing RNA as genetic material. Wendell Stanley produced crystals of TMV in 1935 and found that they contained 5% RNA. But Stanley and others thought TMV was a protein, and that the RNA was either a contaminant, or played a structural role.

A structural role for RNA was reinforced as late as 1955 when Heinz Fraenkel-Conrat separately purified TMV protein and RNA. When he mixed the two components together, they formed infectious, 300 nm rods. When the RNA was omitted, noninfectious aggregates formed. This finding reinforced the belief that RNA helped form virus particles.

TMVThis view changed when Fraenke-Conrat gave his wife, Beatrice Singer, the task of purifying TMV RNA until it had lost all infectivity. To everyone’s surprise she found that TMV RNA itself was infectious, proving in 1957 that it was the viral genetic material. However, RNA also has a structural role in TMV virus particles, as it organizes the capsid protein (yellow in illustration at left) into regularly repeated subunits.

Demonstration of infectivity of RNA from animal viruses soon followed, for mengovirus, a picornavirus, in 1957 and for poliovirus in 1958 (the latter done at my own institution, the College of Physicians and Surgeons of Columbia University!).

By the early 1950s the idea that RNA could be viral genetic material was clearly in the minds of virologists, hence Ephrussi-Taylor’s amusing letter on influenza virus and poliovirus.

*Thanks to @infectiousdose for finding this amazing letter.

TWiV #356: Got viruses?

On episode #356 of the science show This Week in Virology, Stephanie joins the super professors to discuss the gut virome of children with serious malnutrition, caterpillar genes acquired from parasitic wasps, and the effect of adding chemokines to a simian immunodeficiency virus DNA vaccine.

You can find TWiV #356 at www.microbe.tv/twiv.

Wasps do a gain-of-function experiment in caterpillars

parasitic waspParasitic wasps (in the order Hymenoptera) inject their eggs into lepidopteran hosts, where the eggs go through their developmental stages. Along with the eggs, the wasps also deliver viruses carrying genes encoding proteins that inhibit caterpillar immune defenses. Some of these genes are permanently transferred to the lepidopteran host where they have assumed new defensive functions against other viruses.

The viruses that parasitic wasps inject with their eggs, called Bracoviruses, are encoded in the wasp genome. About 100 million years ago a nudivirus genome integrated into the genome of a common wasp ancestor. With time the viral genes became dispersed in the wasp genome. The viruses produced by these wasps today no longer carry capsid coding genes – they are found only in the wasp genome – but only carry genes whose products can modulate lepidopteran defenses. Once in the lepidopteran host, these viruses deliver their genes but no longer form new particles.

An important question is whether wasp Bracoviruses can contribute genes to Lepidoptera – a process called horizontal gene transfer. This possibility would seem remote because the lepidopteran hosts for wasp larvae are dead ends – they die after serving as hosts for wasp development. However, it is possible that some hosts resist killing, or that wasps occasionally inject their eggs and viruses into the wrong host, one that can resist killing.

To answer this question, the genome sequence of Cotesia congregata bracovirus was compared with the genomes of a regular host as well as non-host Lepidoptera. Bracovirus DNA insertions were identified in genomes of the monarch, the silkworm, the beet armyworm and the fall armyworm, but not in the genome of the tobacco hornworm, the usual host of the wasp (C. congregata).

Not only were the Bracovirus sequences found in these varied Lepidoptera, but some appeared to be functional. Two such genes encode a protein that interferes with the replication of baculovirus, a known pathogen of Lepidoptera. This discovery was made in the process of producing the encoded proteins using baculovirus vectors! In other words, viral genes delivered by Hymenopteran wasps were appropriated by the Lepidoptera and used for their defense against a pathogen.

To put it another way, nature has carried out a gain-of-function experiment. Should we impose a moratorium?

The delivery of immunosuppressive viruses by wasps along with their eggs is by all accounts a remarkable story. The appropriation of some of these genes by the wrong hosts should not come as a surprise, yet the finding is nevertheless simply amazing. As long as we keep looking, we will find that the biological world is always full of new revelations.

What does transfection mean?

infectious poliovirus dnaI have always had a problem with the use of the word transfection to mean anything other than the introduction of viral DNA into cells (illustrated for poliovirus). An examination of the origins of the word suggests that such use might be acceptable.

The introduction of foreign DNA into cells is called DNA-mediated transformation to distinguish it from the oncogenic transformation of cells caused by tumor viruses and other insults. The term transfection (transformation-infection) was coined to describe the production of infectious virus after transformation of cells by viral DNA, first demonstrated with bacteriophage lambda. Unfortunately, transfection is now routinely used to describe the introduction of any DNA or RNA into cells.

If you view the English language as a dynamic means of communication that continually evolves and provides words with new meanings, then this incorrect use of transfection probably does not bother you. But scientists must be precise in their use of language, otherwise their ability to communicate will be impaired. This is why the use of transfection to mean anything other than introduction of viral DNA into cells is a bad idea. I do understand that transfection is much easier to write or speak than DNA-mediated transformation, but surely that cannot be an excuse for warping a word’s meaning.

The misuse of transfection bothers me so much, that whenever I see the term, I inspect the usage to see if it is incorrect. Recently after seeing another improper use of the word, I decided to look up its roots. What I found made me reconsider my angst about transfection.

The word ‘trans’ can mean across or through. The word infection, from which the -fection in transfection is derived, comes from the Latin verb inficere: from in (into) + facere (put, do). From this analysis we can determine that transfection means across-put. That is not a bad definition of what transfection has come to mean: put DNA across a membrane into the cell.

I am certainly not a student of etymology, but it seems to me that without knowing it, those who used transfection in the wrong way were actually correct in their usage. I no longer need fret about how transfection is used!

Algal virus associated with altered human cognitive functions

Phycodnaviridae virionMany well-known human viruses, including poliovirus, rabies virus, West Nile virus, can infect cells of the nervous system, leading to alterations in the function of that organ. Could a virus that infects algae also cause human neurological alterations?

Chloroviruses are large DNA-containing viruses that infect unicellular algae called zoochlorellae (pictured: image credit, ViralZone). Unexpectedly, chlorovirus DNA sequences were found in the oropharynx of 40 of 92 individuals (43.5%) who had no known physical or psychiatric illness. The clinical specimens had been obtained as part of a study of cognitive function, and it was possible to determine that presence of chlorovirus DNA was associated with a slight but statistically significant decreased performance in tests for visual motor speed, delayed memory, and attention.

When mice were fed chlorovirus-infected algae, they showed decreased performance in tests of cognitive function, such as recognition memory and sensory-motor gating. Some of these animals developed antibodies against the virus, suggesting that viral replication took place. Furthermore, feeding of chlorovirus to mice was associated with changes in gene expression in the hippocampus, the part of the brain essential for learning, memory, and behavior.

It is not known if the chlorovirus replicates in humans or in mice; only viral nucleic acids were detected. No mention is made of attempts to isolate infectious chloroviruses from humans or mice. The amount of chlorovirus in the oropharynx is not known. However the results of sequence analysis, in which low numbers of sequences were found in each person suggest very low numbers of genomes. Of course, it is possible that virus replication took place some time ago, and its effects linger after replication has subsided.

Chloroviruses are commonly found in inland waters, and the subjects could have acquired the virus via inhalation or drinking contaminated water. It is entirely possible that the virus does not replicate in humans, but is present in the oropharynx as a common environmental contaminant. Many plant and insect virus sequences can be isolated from the human intestinal tract as a consequence of the food we ingest, but there is no evidence that they can replicate at that site. Consequently, chlorovirus might not have any role in the reduced cognitive functions observed in this study. It is possible that exposure to another factor together with chloroviruses, such as heavy metals, is responsible for the observed cognitive differences.

The suggestion that a virus infection might cause subtle cognitive defects is not outlandish. For example, lymphocytic choriomeningitis virus infects rodents congenitally or immediately after birth and establishes a persistent infection of virtually all tissues. These mice show no outward signs of illness, but careful study of infected animals reveals that they are less ‘smart’ than their uninfected peers.

The results are intriguing and warrant more study, including a determination of whether an infectious chlorovirus can be isolated from humans, whether this virus can replicate in human cells in culture, and how they differ from environmental isolates. It would also be important to determine if antibodies to chloroviruses are present in humans, and if they are associated with any diseases. It is too early to conclude that a virus of algae causes altered human neurological functions.

A dancing matrix of viruses

Back in 1974, before it was possible to determine the sequence of a viral genome, before we knew much about the origin of viruses and their ability to move genes from organism to organism, Lewis Thomas wrote the following incredibly prescient words in The Lives of a Cell:

The viruses, instead of being single-minded agents of disease and death, now begin to look more like mobile genes. We live in a dancing matrix of viruses; they dart, rather like bees, from organism to organism, from plant to insect to mammal to me and back again, and into the sea, tugging along pieces of this genome, strings of genes from that, transplanting grafts of DNA, passing around heredity as though at a great party. They may be a mechanism for keeping new, mutant kinds of DNA in the widest circulation among us. If this is true, the odd virus disease, on which we must focus so much of our attention in medicine, may be looked on as an accident, something dropped.

When Thomas wrote these words we knew that bacteriophages could move pieces of DNA from bacterium to bacterium, but we had no idea of the global scale of this movement. We did not know that most viruses could carry genes from cell to cell, nor did we appreciate that viruses could be beneficial. I am amazed by the accuracy of his words written at a time when we knew so little.

TWiV 269: Herpesvirus stops a nuclear attack

On episode #269 of the science show This Week in Virology, the complete TWiV team reviews evidence for sensing of herpesviral DNA in the nucleus by the cell protein IFI16.

You can find TWiV #269 at www.microbe.tv/twiv.

Virologia en Español Clase #7 – Replicación de Virus con Genoma de DNA

En esta sesión discutiremos como los virus con genoma de DNA replican sus ácidos nucleícos. Consideraremos las enzimas y otras proeteínas que participan en la síntesis del DNA viral y los retos que imponen las distintas topologías de los genomas de DNA (DNA linear de cadena sencilla, DNA circular de doble cadena y DNA linear de doble cadena).

Pandoravirus, bigger and unlike anything seen before

pandoravirusThe discovery of the giant Mimivirus and Megavirus amazed virologists (and also many others). Their virions (750 nanometers) and DNA genomes (1,259,000 base pairs) were the biggest ever discovered, shattering the notions that viruses could not be seen with a light microscope, and that viral genomes were smaller than bacterial genomes. Now two even bigger viruses have been discovered, which are physically and genetically unlike any previously known viruses. They have been called Pandoraviruses.

Both new viruses were isolated by culturing environmental samples in the amoeba Acanthamoeba castellaniPandoravirus salinus was isolated from shallow marine sediment in a river at the coast of central Chile, and Pandoravirus dulcis was obtained from mud at the bottom of a freshwater pond near Melbourne, Australia. The P. salinus genome is at least 2.77 megabases in length (there is some uncertainty in the actual length due to the presence of repeated sequences at the ends of the DNA), while the P. dulcis genome is 2.47 megabases in length. The smaller P. dulcis genome is a subset of the P. salinus genome.

These new genomes are twice as large as those of previously described viruses, and bigger than the genomes of intracellular bacteria such as Tremblaya (138,927 base pairs) and Rickettsia (1,111,523 bp), some free living bacteria, and many free living Archaea.

While the huge sizes of the Pandoravirus virion and genomes are amazing, I find three other features of these viruses even more remarkable. The first is their atypical replication cycle. The virions are taken into amoebae by phagocytic vacuoles, and upon fusing with the vacuole membrane, the virion contents are released into the cytoplasm via a pore on the virion apex. Within 2-4 hours the cell nucleus is reorganized, and by 8-10 hours new particles appear where the nucleus once was. Pandoravirus DNA and virions are synthesized and assembled simultaneously, in contrast to eukaryotic DNA viruses and phages which fill pre-formed capsids with DNA. Virions are released by 10-15 hours as the cells lyse.

A second amazing feature is that most of the P. salinus open reading frames encode brand-new proteins. Of the 2,556 putative protein coding sequences in the P. salinus genome, 93% have no recognizable counterparts among known proteins. Some of the genes found in large DNA viruses are present, such as those encoding DNA polymerase and DNA-dependent RNA polymerase, and several amino acyl-tRNA synthetases, like members of the Megaviridae. Curiously, many of the Pandoravirus coding regions contain intervening sequences, which must be removed by RNA splicing. This process is known to occur only in the cell nucleus, suggesting that some Pandoravirus transcription occurs in that organelle. The lack of gene homology leads to authors to conclude that ‘no microorganism closely related to P. salinus has ever been sequenced’.

I am also impressed by what the authors describe as the ‘alien morphological features’ of the virions. The oval-shaped particles are 1 micron in length and 0.5 microns in diameter, easily visible by light microscopy. They are wrapped in a three-layered envelope with a pore at one end of the particle, and resemble nothing that has ever been seen before (see photograph).

How much bigger can viruses get? I don’t know the answer but I would guess even bigger than Pandoraviruses. The membranous Pandoravirus particle could easily accommodate even larger genomes. How big can a virus get and still be a virus? The answer to that question is easy: it is a virus as long as it requires a cell for replication.

These remarkable findings further emphasize the need for scientists to pursue their curiosity, and not only work on problems of obvious medical relevance. As the authors write,

This work is a reminder that our census of the microbial diversity is far from comprehensive and that some important clues about the fundamental nature of the relationship between the viral and the cellular world might still lie within unexplored environments.

Continuing their playful naming of giant viruses, the authors note that the name Pandoravirus reflects their ‘lack of similarity with previously described microorganisms and the surprises expected from their future study’.