Have a methyl with your viral RNA

N6-MethyladenosineChemical 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  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 of 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.

Animal viruses with separately packaged RNA segments

dose-response-plaque-assayThere are many examples of viruses with segmented genomes – like influenza viruses – but these genomes segments are packaged in  one virus particle. Sometimes 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 of  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.

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.

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!

Viroids, infectious agents that encode no proteins

potato spindle tuber viroidGenomes of non-defective viruses range in size from 2,400,000 bp of dsDNA (Pandoravirus salinus) to 1,759 bp of ssDNA (porcine circovirus). Are even smaller viral genomes possible? The subviral agents called viroids provide an answer to this question.

Viroids, the smallest known pathogens, are naked, circular, single-stranded RNA molecules that do not encode protein yet replicate autonomously when introduced into host plants. Potato spindle tuber viroid, discovered in 1971, is the prototype; 29 other viroids have since been discovered ranging in length from 120 to 475 nucleotides. Viroids only infect plants; some cause economically important diseases of crop plants, while others appear to be benign. Two examples of economically important viroids are coconut cadang-cadang viroid (which causes a lethal infection of coconut palms) and apple scar skin viroid (which causes an infection that results in visually unappealing apples).

The 30 known viroids have been classified in two families. Members of the Pospiviroidae, named for potato spindle tuber viroid, have a rod-like secondary structure with small single stranded regions, a central conserved region, and replicate in the nucleus (illustrated; click to enlarge; figure credit). The Avsunviroidae, named for avocado sunblotch viroid, have both rod-like and branched regions, but lack a central conserved region and replicate in chloroplasts. In contrast to the Pospiviroidae, the latter RNA molecules are functional ribozymes, and this activity is essential for replication.

There is no evidence that viroids encode proteins or mRNA. Unlike viruses, which are parasites of host translation machinery, viroids are parasites of cellular transcription proteins: they depend on cellular RNA polymerase for replication. Such polymerases normally recognize DNA templates, but can copy viroid RNAs.

In plants infected with members of the Pospiviroidae, viroid RNA is imported into the nucleus, and copied by plant DNA-dependent RNA polymerase II. The viroid is copied by a rolling circle mechanism that produces complementary linear, concatameric, RNAs. These are copied again to produce concatameric, linear molecules, which are cleaved by the host enzyme RNAse III. Their ends are joined by a host enzyme to form circles.

In plants infected with members of the Avsunviroidae, viroid RNA is imported into the chloroplast, and complementary concatameric RNAs are produced by chloroplast DNA-dependent RNA polymerase. Cleavage of these molecules is carried out by a ribozyme, an enzyme encoded in the viroid RNA.

After replication, viroid progeny exit the nucleus or chloroplast and move to adjacent cells through plasmodesmata, and can travel systemically via the phloem to infect other cells. Viroids enter the pollen and ovule, from where they are transmitted to the seed. When the seed germinates, the new plant becomes infected. Viroids can also be transmitted among plants by contaminated farm machinery and insects.

Symptoms of viroid infection in plants include stunting of growth, deformation of leaves and fruit, stem necrosis, and death. Because viroids do not produce mRNAs, it was first proposed that disease must be a consequence of viroid RNA binding to host proteins or nucleic acids.  The discovery of RNA silencing in plants lead to the hypothesis that small interfering RNAs derived from viroid RNAs guide silencing of host genes, leading to induction of disease. In support of this hypothesis, peach latent mosaic viroid small RNAs have been identified that silence chloroplast heat shock protein 90, which correlates with disease symptoms. The different disease patterns caused by viroids in their hosts might all have in common an origin in RNA silencing.

Our current understanding is that the disease-causing viroids were transferred from wild plants used for breeding modern crops. The widespread prevalence of these agents can be traced to the use of genetically identical plants (monoculture), worldwide distribution of breeding lines, and mechanical transmission by contaminated farm machinery. As a consequence, these unusual pathogens now occupy niches around the planet that never before were available to them.

The origin of viroids remains an enigma, but it has been proposed that they are relics from the RNA world, which is thought to have been populated only by non-coding RNA molecules that catalysed their own synthesis. Viroids have properties that make them candidates for survivors of the RNA world: small genome size (to avoid error catastrophe caused by error-prone replication), high G+C content (for greater thermodynamic stability), circular genomes (to avoid the need for mechanisms to prevent loss of information at the ends of linear genomes), no protein content, and the presence of a ribozyme, a fingerprint of the RNA world. Today’s viroids can no longer self-replicate, possibly having lost that function when they became parasites of plants. What began as a search for virus-like agents that cause disease in plants has lead to new insights into the evolution of life.

Virologia en Español clase #8 – Transcripción de genomas de DNA

En esta clase revisaremos como se sintetizan los pre-RNA mensajeros a partir de sus templados de DNA, y como los pre-mRNA se convierten a mRNAs maduros mediante splicing, poliadenilación, y su exportación del núcleo al citoplasma celular.


A DNA virus with the capsid of an RNA virus

Boiling Springs Lake Lassen NPViral genomes are unusual because they can be based on RNA or DNA, in contrast to all cellular life forms, which have DNA as their genetic information. An unusual new virus has been discovered that appears to have sequences from both an RNA and a DNA virus.

The new virus was identified during a study of viral diversity in an extreme environment, Boiling Spring Lake. You would never want to swim there: it is acidic (pH 2.5) and hot (52° − 95° C). But the lake is not devoid of living things: it is inhabited by various bacteria, Archaea, and unicellular eukaryotes. Where there is life, there are viruses, which leads us to an expedition to determine what kinds of viruses can be found in Boiling Spring Lake.

To answer this question, Goeff Diemer and Kenneth Stedman sequenced viral DNA extracted from purified viral particles from Boiling Spring Lake water. Their analyses revealed the presence of a virus with a circular, single-stranded DNA genome similar to that found in members of the Circoviridae (this virus family includes porcine circovirus and chicken anemia virus). What surprised the investigators was that the gene encoding the viral capsid protein was similar to that from viruses with single-stranded RNA genomes, including viruses that infect plants (Tombusviridae) or fungi. The authors call it ‘RNA-DNA hybrid virus’, or RDHV. The host of RDHV is unknown but could be one of the eukaryotes that inhabit Boiling Spring Lake.

RDHV probably arose when a circovirus acquired the capsid protein of an RNA virus by DNA recombination. This event likely occurred in a cell infected with both viruses. A cellular reverse transcriptase might have converted the circovirus RNA genome to DNA to allow recombination to occur. RDHV is unusual because genetic exchanges among viruses are restricted to those with similar genome types.

To determine if RDHV is an oddity, the authors searched the database of DNA sequences obtained from the Global Ocean Survey. They found three RDHV-like genomes, indicating that these viruses exist in the ocean. Whether they are present elsewhere is a question that should certainly be answered. It is important to determine whether recombination between RNA and DNA viruses is a common means of gene exchange, or whether it is a rarity.

The discovery of RDHV could have implications for viral evolution. It has been suggested that the first organisms that evolved on earth were based on RNA molecules with coding and catalytic capabilities. Later, DNA based life evolved, and today both DNA based and RNA based organisms co-exist. Viruses like RDHV could have emerged during the transition from an RNA to a DNA world, when a new DNA virus captured the gene encoding an RNA virus capsid. In other words, RNA genes that had already evolved were not discarded but appropriated by DNA viruses. This scenario would have required some mechanism for converting RNA into DNA (reverse transcriptases?). The finding of RDHV-like viruses in the ocean suggests that a common ancestor emerged some time ago which diversified into different environments. More RDHV-like viruses must be isolated and studied before we can determine whether or not these viruses are very old, and to deduce their implications for viral evolution.

Diemer GS, Stedman KM. 2012. A novel virus discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses. Biology Direct 7:13.

TWiV 180: Throwing IFIT at flu and holding a miR to HCV

On episode #180 of the science show This Week in Virology, Vincent, Alan, and Rich review association of an interferon-induced protein with severe influenza, and stabilization of HCV RNA by a microRNA.

You can find TWiV #180 at www.microbe.tv/twiv.

Har Gobind Khorana, master decoder

genetic codeMost students of elementary biology will have seen the table at left that depicts the genetic code. HG Khorana was one of several scientists who determined, in the 1960s, the amino acids specified by each three-letter combination of bases. As long as I have been at Columbia University I have had a copy of this table on my office wall.

Khorana reminisces how the finding that genes are nucleic acids set the stage for his work on decoding:

While it is always difficult, perhaps impossible, to determine or clearly define the starting point in any area of science, the idea that genes make proteins was an important step and this concept was brought into sharp focus by the specific one gene-one enzyme hypothesis of Beadle and Tatum. The field of biochemical genetics was thus born. The next step was taken when it was established that genes are nucleic acids. The transformation experiments of Avery and coworkers followed by the bacteriophage experiments of Hershey and Chase established this for DNA and the work with TMV-RNA a few years later established the same for RNA. By the early 1950’s it was, therefore, clear that genes are nucleic acids and that nucleic acids direct protein synthesis, the direct involvement of RNA in this process being suggested by the early work of Caspersson and of Brachet.

The first triplet to be decoded was UUU, which specifies the amino acid phenylalanine. This work was done by Nirenberg who found that an RNA consisting of repeating U residues could be translated into a protein containing only phenylalanine. Codons for lysine (AAA) and proline (CCC) were similarly discovered using RNAs containing only A or C. Khorana used both enzymatic and chemical synthesis of oligonucleotides to decipher much of the remaining code. A good account of his work can be found in his Nobel lecture (pdf).

Khorana, together with Robert Holley (structure of tRNA) and Marshall Nirenberg, received the Nobel Prize in Physiology or Medicine in 1968 for their work on deciphering the genetic code.

Virology lecture #6: RNA-directed RNA synthesis

Download: .wmv (324 MB) | .mp4 (76 MB)

Visit the virology W3310 home page for a complete list of course resources.