Posts Tagged ‘ssDNA’

Setting your virus free

7 August, 2012

I was reminded, as I walked in my garden in the Cape Town late afternoon sun a short while ago, of a Master’s degree project I had started with a very bright young person.  A young person who didn’t finish, because she abandoned her degree in the interests of finding herself – and subsequently got into computer-based education some 16 years ago, but that’s another story.

I have written about her previously, as it happens: she is the “Dr” Jacobson in this story, written about a year after her Honours essay on Emerging Viruses became the most authoritative source in the world on Ebola virus that was available electronically – and the Kikwit Ebola outbreak that occurred soon afterwards caused the world to go frantically looking for information.

Sadly for her, she could not work on Ebola for her Master’s, so I gave her what I thought was the next best thing: a project on making a replicating DNA vector system out of Abutilon mosaic virus (AbMV), a two-component ssDNA begomovirus.  The project started in the easiest way imaginable: she went to the local plant nursery, and bought a variegated Abutilon striatum in a pot, and planted it in our Departmental plant room.

Abutilon leaves and flower

AbMV in what is now an ornamental abutilon produces very striking symptoms, which accounts for the popularity of the plant, and its spread across much of the world – by cuttings, mainly.  This is fortunate, as in most cases the virus has lost its natural mode of transmission, which is only via whiteflies (Bemisia tabaci).  Thus, by fortuitous accident, a virus that is  effectively crippled is now spread far beyond its point of origin in South America, purely by human intervention.

Be this as it may, our mission was to harness the fact that AbMV maintains itself as an episome for the lifetime of a plant by making it into an expression vector for plant-made vaccines.  Kenneth Palmer in my lab had already done similar work with Maize streak virus; however, maize was not really a usable host because it is an annual and was hard to infect and the vector did not spread.  It was also not usable in dicot hosts, so we settled on AbMV as being available in our and many other back yards.

We did not get far: while Alison was really bright, by this time she had discovered that science really wasn’t for her, and made essentially no progress beyond cloning a B genome and getting some sequence out of it.  She left to find more fulfilling things to do, and her experimental material continued to grow in the plant room – and gather red spider mites.  I still have the badge off her labcoat, incidentally: I couldn’t let it go; it was and is the only Led Zeppelin logo I have ever seen on the standard white coat.

This is where we get to the title of this post. In 1996 or so, I took the by-now largish plant in its pot back home, and set it free: I planted it in my garden.  It eventually developed into a large bush, easily 3 x 2 metres wide and tall – and has just been cut back, after some 16 years, to allow it to redevelop.  I get a little kick out of seeing civilians step nervously away from it, after I have walked them up to it, and say: “And this is the biggest virus you will ever meet”.  Let’s see you do THAT, Ebola virologists!

Oh, it isn’t entirely free: we sampled it again a couple of years ago when the fearsomely efficient geminivirus-hunting crew that grew out of my lab wanted samples to test their then-new phi29 rolling circle amplification chops on.  We could still only get a B genome out of it, and one that was 10% different from any other published AbMV – so maybe there’s still a story there.

But all it has to do now is keep on growing.  And look beautiful.

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-)