Did you know that the innate immune DNA sensor TLR9 is on the membrane of red blood cells? I didn’t know that. To learn about why it’s there, listen to Immune episode #50. In that episode we review evidence that toll-like receptor 9 on the surface of red blood cells binds DNA, leading to uptake by macrophages and innate immune activation.
A 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 formsÂ during viral infection of bacteriaÂ 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 translationÂ 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 Â 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.Â According to one hypothesis, self-replicating, virus-like nucleic acids might have first appeared on Earth, followed by cellsÂ without a nucleus.Â Was the nucleus a viral invention?
Today it is wellÂ known that viruses may containÂ DNA (poxvirus, mimivirus) or RNA (influenza virus, Zika virus), but for many years it was thought that genomes were only made ofÂ DNA. The surprise at finding only RNA in a virusÂ isÂ 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 thatÂ 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 structureÂ of DNA in 1953, and a few years later published the Central Dogma, which suggested thatÂ 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, Â comes the note in 1953 fromÂ 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 purifiedÂ 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.
This 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 ofÂ RNA from animal viruses soon followed, forÂ 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 clearlyÂ in the minds of virologists, henceÂ Ephrussi-Taylor’s amusing letter on influenza virus and poliovirus.
*Thanks to @infectiousdose for finding this amazing letter.
On episode #356 of the science show This Week in Virology,Â 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.
Parasitic wasps (in the order Hymenoptera) inject their eggs into lepidopteran hosts, where theÂ eggs go throughÂ their developmental stages. Along with the eggs, the wasps also deliver viruses carrying genes encoding proteins thatÂ 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.
I have always had a problem with the use of the word transfectionÂ 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 transfectionÂ (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 wordâ€™s meaning.
The misuse of transfection bothers me soÂ much, that whenever I see the term, I inspect the usage to see if it is incorrect. Recently after seeing another improper useÂ of the word, I decided to look up its roots. What I found made me reconsider my angst about transfection.
The wordÂ â€˜transâ€™ can mean across or through. The word infection, fromÂ which the -fection in transfection is derived, comes from the Latin verbÂ inficere: fromÂ inÂ (into) +Â 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 withoutÂ knowingÂ it, those who used transfection in the wrong way were actually correct in their usage. I no longer need fret aboutÂ how transfection is used!