Posts Tagged ‘rolling circle’

Silence(d) is Golden (mosaic)…

12 October, 2011

Geminivirus particle: characteristic doubled icosahedron containing a single ssDNA (courtesy Russell Kightley)

About that title…I read in my Nature News on the iPad about the use of siRNA in transgenic beans to silence expression of the Bean golden mosaic begomovirus, and I irresistibly thought of this…B-)

To serious matters – said article reported the following:

“Brazilian scientists roll out a transgenic pinto bean (Phaseolus vulgaris) engineered to fend off one of the crop’s most devastating enemies: the golden mosaic virus. Approved on 15 September by the Brazilian National Technical Commission on Biosafety (CTNBio), the transgenic bean uses RNA interference to shut down replication of the virus [reported originally in Mol Plant Microbe Interac in 2007].”

This paper reported the following:

“…we explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generate highly resistant transgenic common bean plants. Eighteen transgenic common bean lines were obtained with an intron-hairpin construction to induce post-transcriptional gene silencing against the AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of the plants were free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant during the whole plant life cycle) and at a very early stage of plant development. “

OK, some background: Bean golden mosaic virus (BGMV) is a begomovirus, a representative of the largest genus of the Geminiviridae, and one of the more devastating viral plant pathogens on the planet.  It is a single-stranded circular DNA virus with a very distinct particle morphology, which replicates its genome by a rolling circle mechanism shared by all geminiviruses, nanoviruses, circoviruses, microviruses and pretty much any other ssDNA virus, as well as some plasmids.

RNA silencing – once known as post-transcriptional gene silencing, before the field was usurped by non-plant virologists – is a natural mechanism used by plants in particular as an adaptive immune response to plant viruses, as well as to control gene expression.  It is a complicated process, involving the formation of double-stranded RNAs from complementary sequences, transcribed from DNA or RNA genomes, which are then chopped up into shorter 21-25 base-length sequences.  These small interfering (si) RNAs are dissociated, and are free to bind to complementary sequences in the plant cell cytoplasm – and target them for degradation by a particular set of enzymes.  This happens frequently in transgenic plants, where the desired over-expression of a particular gene may be frustrated by the plant promptly silencing it.  It is also part of an arms race between plant viruses and plants, with nearly all plant viruses demonstrating some ability to interfere with siRNA silencing.

Geminiviruses are no exception: a number of papers have explored silencing suppression by geminiviruses, with a review by Dave Bisaro prominent among them.  Who is also famous for singing “Born to be Wild” in a Spanish karaoke bar in 1994 with a number of other geminivirologists, who called themselves “Subgroup IV” – but I digress.

It is interesting, then, that one can make transgenic plants expressing siRNA specific for a geminivirus gene – and get silencing of viral expression, and effective immunity to the virus: this would seem to have potential for a deathmatch, with the plant trying to silence virus-coded RNA, while the virus tries to suppress RNA silencing by the plant…as well as the fact that it is a DNA virus, and silencing is mediated at the level of cytoplasmic RNA.

But it obviously works – and probably because the siRNA is being expressed constitutively, meaning the virus infecting the first cell(s) gets shut down before it has a chance to get expression going.  The choice of gene – the “AC1″ or Rep – is also important, as expression of mRNA from this is at a very low level, and it is crucial for virus genome replication.  This means that shutting it down stops any DNA replication from occurring.

So Viva! Brasil, Viva! as we South African are fond of saying.  Southern hemisphere rules geminivirus resistance, OK…because we have more than a passing interest in the same phenomenon…B-)

Virus Origins II

28 September, 2011

I have updated the blog on virus origins quite considerably – new pictures, more detail, more speculation!

Pathways on information flow for RNA viruses

What are you looking for?

18 April, 2011

It is quite educational sometimes, to look and see what it is people are looking for, when they end up on ViroBlogy.  Here are the queries over the course of the last month:

Search Views
ebola 577
ebola virus 64
rolling circle 44
how did viruses evolve 29
mimivirus 21
despair posters 19
flaviviridae 17
lassa fever 14
bacterial dna replication 11
ebola virus patients 11
viroblogy 9
where did viruses evolve from 9
ebola pictures 9
rift valley fever 9
what did viruses evolve from 9
geminivirus 8
west nile virus structure 8
ebola patient 8
where h1n1 came from 7
ebola virus pictures 7

I count 6 mentions of Ebola, 4 of virus evolution generally…and a gratifyingly high number of queries on rolling circle replication, given our lab’s interest in it.  Of course, I have had long had an obsession with Ebola and similar nasties, and have been deeply interested in the evolution of viruses ever since I started studying them, so it is good to see others share my interests!

So in the spirit of giving people what they want, look for blog posts with titles like “The evolution of viruses using rolling circle replication: its complete non-relevance to Ebola and H1N1 influenza viruses”.  But seriously – I will be putting up some more posts around the most popular subject areas, if only to help make sure that The Truth Gets Out There.

Ed Rybicki

Virus origins: from what did viruses evolve or how did they initially arise?

19 March, 2008

This was originally written as an Answer to a Question posted to Scientific American Online; however, as what they published was considerably shorter and simpler than what I wrote, I shall post the [now updated] original here.

The answer to this question is not simple, because, while viruses all share the characteristics of being obligate intracellular parasites which use host cell machinery to make their components which then self-assemble to make particles which contain their genomes, they most definitely do not have a single origin, and indeed their origins may be spread out over a considerable period of geological and evolutionary time.

Viruses infect all types of cellular organisms, from Bacteria through Archaea to Eukarya; from E. coli to mushrooms; from amoebae to human beings – and virus particles may even be the single most abundant and varied organisms on the planet, given their abundance in all the waters of all the seas of planet Earth.  Given this diversity and abundance, and the propensity of viruses to swap and share successful modules between very different lineages and to pick up bits of genome from their hosts, it is very difficult to speculate sensibly on their deep origins – but I shall outline some of the probable evolutionary scenarios.

It is generally accepted that many viruses have their origins as “escapees” from cells; rogue bits of nucleic acid that have taken the autonomy already characteristic of certain cellular genome components to a new level.  Simple RNA viruses are a good example of these: their genetic structure is far too simple for them to be degenerate cells; indeed, many resemble renegade messenger RNAs in their simplicity.

RNA virus supergroups and RdRp and CP cassettes

What they have in common is a strategy which involves use of a virus-encoded RNA-dependent RNA polymerase (RdRp) or replicase to replicate RNA genomes – a process which does not occur in cells, although most eukaryotes so far investigated do have RdRp-like enzymes involved in regulation of gene expression and resistance to viruses.  The surmise is that in some instances, an RdRp-encoding element could have became autonomous – or independent of DNA – by encoding its own replicase, and then acquired structural protein-encoding sequences by recombination, to become wholly autonomous and potentially infectious.

A useful example is the viruses sometimes referred to as the”Picornavirus-like” and “Sindbis virus-like” supergroups of ssRNA+ viruses, respectively.  These two sets of viruses can be neatly divided into two groups according to their RdRp affinities, which determine how they replicate.  However, they can also be divided according to their capsid protein affinities, which is where it is obvious that the phenomenon the late Rob Goldbach termed “cassette evolution” has occurred: some viruses that are relatively closely related in terms of RdRp and other non-structural protein sequences have completely different capsid proteins and particle morphologies, due to acquisition by the same RdRp module of different structural protein modules.

Given the very significant diversity in these sorts of viruses, it is quite possible that this has happened a number of times in the evolution of cellular organisms on this planet – and that some single-stranded RNA viruses like bacterial RNA viruses or bacteriophages and some plant viruses (like Tobacco mosaic virus, TMV) may be very ancient indeed.

However, other ssRNA viruses – such as the negative sense mononegaviruses, Order Mononegavirales, which group includes Ebola, measles and mumps and rabies viruses – may be evolutionarily much younger.  In this latter case, the viruses all have the same basic genome with genes in the same order and helical nucleocapsids within differently-shaped enveloped particles.  Their host ranges also indicate that they originated in insects: the ones with more than one phylum of host either infect vertebrates and insects or plants and insects, while some infect insects only, or only vertebrates – indicating a possible evolutionary origin in insects, and a subsequent evolutionary divergence in them and in their feeding targets.

HIV: a retrovirus

The ssRNA retroviruses are another good example of possible cell-derived viruses, as many of these have a very similar genetic structure to elements which appear to be integral parts of cell genomes – termed retrotransposons -  and share the peculiar property of replicating their genomes via a pathway which goes from single-stranded RNA through double-stranded DNA (reverse transcription) and back again, and yet have become infectious.  They can go full circle, incidentally, by permanently becoming part of the cell genome by insertion into germ-line cells – so that they are then inherited as “endogenous retroviruses“, which can be used as evolutionary markers for species divergence.

The Retroid Cycle

Indeed, there is a whole extended family of reverse-transcribing mobile genetic elements in organisms ranging from bacteria all the way through to plants, insects and vertebrates, indicating a very ancient evolutionary origin indeed – and which includes two completely different groups of double-standed DNA viruses, the vertebrate-infecting hepadnaviruses or hepatitis B virus-like group, and the plant-infecting badna- and caulimoviruses.  All of these cellular elements and viruses have in common a “reverse transcriptase” or RNA-dependent DNA polymerase, which may in fact be an evolutionary link back to the postulated “RNA world” at the dawn of evolutionary history, when the only extant genomes were composed of RNA, and probably double-stranded RNA.  Thus, a part of what could be a very primitive machinery indeed has survived into very different nucleic acid lineages, some viral and many wholly cellular in nature, from bacteria through to higher eukaryotes.

The possibility that certain non-retro RNA viruses can actually insert bits of themselves by obscure mechanisms into host cell genomes – and afford them protection against future infection – complicates the issue rather, by reversing the probable flow of genetic material!

rolling circle replicationThere are also obvious similarities in mode of replication between a family of elements which include bacterial plasmids, bacterial single-strand DNA viruses, and viruses of eukaryotes which include geminiviruses and nanoviruses of plants, parvoviruses of insects and vertebrates, and circoviruses and anelloviruses of vertebrates.

Geminivirus particle

These agents all share a “rolling circle” DNA replication mechanism, with replication-associated proteins and DNA sequence motifs that appear similar enough to be evolutionarily related – and again demonstrate a continuum from the cell-associated and cell-dependent plasmids through to the completely autonomous agents such as relatively simple bacterial and eukaryote viruses.

Mimivirus particle, showing basic structure

However, there are a significant number of viruses with large DNA genomes for which an origin as cell-derived subcomponents is not as obvious.  In fact, the largest viruses yet discovered – mimiviruses, with a genome size of greater than 1 million base pairs of DNA – have genomes which are larger and more complex than those of obligately parasitic bacteria such as Mycoplasma genitalium (around 0.5 million), despite their sharing the life habits of tiny viruses like canine parvovirus (0.005 million, or 5000 bases).  In fact, it is a striking fact that the largest viral DNA genomes so far characterised seem to infect primitive eukaryotes such as amoebae and simple marine algae – and they and other large DNA viruses like pox- and herpesviruses seem to be related to cellular DNA sequences only at a level close to the base of the “tree of life”.

Variola virus, the agent of smallpox. Image courtesy Russell Kightley Media.

This indicates a very ancient origin or set of origins for these viruses, which may conceivably have been as obligately parasitic cellular lifeforms which then made the final adaptation to the “virus lifestyle”.  However, their actual origin could be in an even more complex interaction with early cellular lifeforms, given that viruses may well be responsible for very significant episodes of evolutionary change in cellular life, all the way from the origin of eukaryotes through to the much more recent evolution of placental mammals.  In fact, there is informed speculation as to the possibility of viruses having significantly influenced the evolution of eukaryotes as a cognate group of organisms, including the possibility that a large DNA virus may have been the first cellular nucleus.

In summary, viruses are as much a concept as a unitary entity: all viruses have in common, given their polyphyletic origins, is a base-level strategy for replicating their genomes.  Otherwise, their origins are possibly as varied as their genomes, and may remain forever obscure.

I am indebted to Russell Kightley for use of his excellent virus images.

Updated 28th September 2011

MicrobiologyBytes Archive

14 December, 2007

Before I established this site, I posted a number of guest blogs to do with viruses on Alan Cann’s very wonderful MicrobiologyBytes site. Here are links to all the virus-related ones.

Maybe Not Quite The End

Posted on January 15, 2008
Review of a paper describing the receptor for the H5N1 HA protein

Given the current scare over H5N1 influenza virus in swans in the UK, it is possibly timely to recall that I wrote a little while ago in MicrobiologyBytes about how easy it appeared to be for […]

Bandicoot Blues

Posted on November 30, 2007
Description of a unique newly-described virus that looks like a chimaera of a papillomavirus and a polyomavirus

Now that the dust has begun to settle after the launch of Merck’s much-hyped Gardasil genital papillomavirus vaccine – discussed in MicrobiologyBytes here and here – people are turning again to looking at the natural history […]

Hurting rather than helping?

Posted on November 21, 2007
Some news on the failure of the Merck Adenovirus 5-vectored HIV vaccine

It should not have escaped the eye of the interested bystander that there has been a most unfortunate and premature end to a HIV vaccine trial recently – and that something that had been tested as […]

A Deeper Meaning

Posted on November 10, 2007
Some microbiology-related poetry….

I inadvertently became a published literary critic a little while ago. A long-time English Department colleague asked me for some help interpreting the collected works of possibly the most important modern poet from South Africa, and […]

Don’t look now, they’re in your genes

Posted on September 14, 2007
Description of natural insertions of virus gene fragments into a variety of organisms and how they elicit pathogen-derived resistance

And they’re protecting you! If you’re an insect, that is. Or possibly a plant.
In a remarkable convergence of news, an Israeli group led by Ilan Sela described how Israeli acute paralysis virus, which is implicated in […]

To bee or not to bee

Posted on September 11, 2007
News of how a single virus is suspected in the causation of “colony collapse disorder” of bee hives in the USA

A major recent mystery in US agriculture has been the phenomenon of “colony collapse disorder” (CCD) in honey bees. […]

This is the End

Posted on August 29, 2007
H5N1 highly pathogenic avian influenza virus mutates…

This is the End. Or the beginning of the end. Or possibly, the end of the beginning?
To misquote the immortal Bill Shankly: “It’s not a matter of life and death: it’s much more important than that”.
Having […]

Rolling down the road

Posted on August 27, 2007
Musings on rolling circle replication in viruses

In my idle moments (alas, too few these days!) I often try to think up lists of rock songs with a virus theme: you know, like “Cucumo” by the Beech Boys… “I got them ol’ burnin’, […]

Rooting the tree

Posted on August 3, 2007
News on inferring “ancestor sequences” for HIV to help make broadly effective vaccines

While fossilized viruses have never been found, we can often infer probable lines of evolutionary descent by analysis of extant genomic sequences. This sort of molecular phylogenetic approach has thrown up all sorts of interesting […]

It’s Life, Jim, but not as we know it…

Posted on July 24, 2007
Exploring what it means to be “alive”

Which could well apply to viruses, my very own favourite organisms – after all, they don’t respire, grow, excrete or any of those other good things […]

A feeling for the molechism*

Posted on June 26, 2007
Musings on what viruses are.

I think it’s permissible, after working on your favourite virus for over 20 years, to develop some sort of feeling for it: you know, the kind of insight that isn’t […]

Plus ça change, plus c’est … le same Web, only better?

Posted on June 8, 2007
A personal history of teaching Virology via the Web.

My, how things do change… I found myself reflecting, while I was looking over the detritus on our Web server of some 13 years of posting pages on the Web. “Orphan” pages, unconnected […]


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