The development of high throughput nucleic acid sequencing tools has rapidly increased the pace of virus discovery in the past 20 years. Yet in that time, while the largest DNA genomes have increased by nearly ten times, the largest known RNA viral genome has only increase in size by a tenth. This situation has now changed with the discovery of new RNA viruses of planarians and mollusks.
Vincent, Kathy and Rich travel to ASM Microbe 2018 in Atlanta where they speak with Stacy Horner and Ken Stapleford about their careers and their research.
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Chemical modification of RNA by the addition of methyl groups is known to alter gene expression without changing the nucleotide sequence. The addition of a methyl group to adenosine has been found to regulate gene expression of animal viruses, and most recently of plant viruses.
The illustration shows a methyl (CH3-) group added to the nitrogen Â that is attached to the #6 carbon of the purine base adenine. The entire molecule, with the ribose, is called N6-methyladenosine (m6A). Methylation of adenosine is carried out by enzymes that bind the RNA in the cell cytoplasm.
The m6A modification is found in multiple RNAs of most eukaryotes. It has also been found in the genome of RNA animal viruses. The modification is added to RNAs by a multi-protein enzyme complex, and is removed by demethylases. Silencing of the methylases decreases HIV-1 replication, while depletion of demethylases has the opposite effect. The replication of other viruses, including hepatitis C virus and Zika virus, is also regulated by m6A modification, but the details differ. For example, m6A negatively affects the replication of the flaviviruses hepatitis C virus and Zika virus.
Methylation of adenosine has been recently shown to modulate the replication of plant viruses. The RNA genomes of alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV) were found to contain m6A. An m6A demethylase was identified in Arabidopsis thaliana, a small flowering plant commonly used in research. This demethylase protein bound the capsid protein of AMV but not of CMV. Elimination of the demethylase from Arabidopsis reduced the replication of AMV but not CMV. These results show that m6A methylation negatively regulates the replication of AMV. Binding of the AMV capsid protein to the m6A demethylase might be a mechanism for ensuring that the enzyme demethylates viral RNA, allowing for efficient viral replication.
While it is clear that m6A regulates the replication of RNA viruses, the mechanisms involved are not well understood. Methylation of adenosine is likely to affect multiple functions, including the structure, celllular localization, splicing, stability, and translation ofÂ viral RNA (link to review). As m6A is also found in cellular RNAs, studies of its effect on viral processes is likely to provide insight into its role in cellular biology.
There are many examples of viruses with segmented genomes – like influenza viruses – but these genomes segments are packaged in Â one virus particle. SometimesÂ the genome segments are separately packaged in virus particles. Such multicomponent viruses are commonly found to infect plants and fungi, but only recently have examples of such viruses that infect animals been discovered (paper link).
Sequence analysis of viruses isolated from Culex mosquitoes in Central and South American Countries revealed six new viruses with segmented RNA genomes, which was confirmed by gel electrophoresis of RNA extracted from virus particles.
Some of the virus isolates appear to lack the fifth RNA segment, and the results of RNA transfection experiments indicated that this RNA is not needed for viral infectivity.
RNA viruses with segmented genomes are common, but in this case, the surprise came when it was found that the dose-response curve of infection for these viruses was not linear. In other words, one virus particle was not sufficient to infect a cell (illustrated). In this case, between 3 and 4 particles were needed to establish an infection. These findings indicate that the viral RNA segments are separately packaged, and must enter a cell together to initiation infection.
These novel viruses, called Guaico Culex virus (GCXV) are distantly related to flaviviruses, a family of non-segmented, + strand RNA viruses. They are part of a clade of RNA viruses with segmented genomes called the Jingmenvirus, which includes a novel tick-borne virus isolated in China (previously discussed on this blog), and a variant isolated from a red colobus monkey in Uganda. These viruses are also likely to have genomes that are separately packaged.
An interesting question is to identify the selection that lead to the emergence ofÂ multicomponent viruses that require multiple particles to initiate an infection. Perhaps transmission of these types of viruses by insect vectors facilitates the introduction of multiple virus particles into a cell. How such viral genomes emerged and persisted remains a mystery that might be solved by the analysis of other viruses with similar genome architectures.
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.
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!