Synthesizing a Virus

Synthesizing Genomes

Viruses can be viewed both as chemicals and as "living" entities. They can be crystallized, and their empirical formulas and structures can be determined, yet they can replicate naturally in organisms from bacteria to humans.

Poliovirus is a small icosahedral virus consisting of

60 copies each of five capsid proteins, forming an exterior "shell"
One positive-sense single-stranded RNA genome of about 7.5 kilobases
The sequence, the map of the genome, and the crystal structure have been known for almost 30 years

The Capsid of the Sabin Strain of the Poliovirus

The life cycle is simple:

After the virus penetrates a cell, the RNA is translated in the ribosome into a single long protein
Two viral proteases chop the long protein chain into functional proteins
A viral RNA polymerase then transcribes the viral RNA into negative-sense (complementary) copies
These copies serve as templates for the synthesis of new positive-sense genomes
These can serve as mRNA for more copies of the viral proteins, or be encapsulated to form a new virus particle
Eventually, the cell bursts and releases the new virus particles to infect other cells

Wimmer and co-workers [Science, 2002, 297, 1016] carried out the chemical synthesis of the virus cDNA (that is, a strand of DNA complementary to the RNA of the virus)

The resulting DNA was inserted into a plasmid vector and then transcribed by an RNA polymerase.

The resulting viral RNA translated and replicated in a cell free extract, producing infectious polio virus.

When added to a culture of HeLa cells, this virus produced an infection in all ways identical to the natural viral infection.
[HeLa is a line of tumor cells derived from a uterine cancer suffered by Henrietta Lacks; read about this in "The Immortal Life of Henrietta Lacks", by Rebecca Skloot.]
Tumpey et al. [Science, 2005, 310, 77; Nature Biotechnol., 2009, 27, 1163] have synthesized the 1918 "Spanish" flu virus, that killed as many as 36 million people worldwide. A major reason for doing this was to understand what made this virus so lethal; in addition, they wished to learn its evolutionary history.
The 1918 virus was an H1N1 type.
- H1 is the particular mutant of the hemagglutinin protein, which causes erythrocytes to clump
- N1 likewise specifies a particular mutant, this time of the neuramidinase protein, which attaches to, and cleaves the oligosaccharides displayed on the cell surface
The Neuramindinase from the 1918 influenza virus
complexed to the antiviral Relenza (3b7e) An Avian H1N1 Hemagglutinin (3hto)

A good discussion of how the hemagglutinin and neuramidinase work: J. Bio. Chem., 2010, 285, 28403. A review of synthetic viruses: Wimmer et al., Nature Biotechnol., 2009, 27, 1163.
Viruses may not actually be alive, but bacteria certainly are.
The bacterium Mycoplasma genitalium is believed to have the smallest genome of any free living organism: 582,790 base pairs, which code for 482 proteins.
Smith and Venter [Proc. Nat. Acad. Sci., 2006, 103, 425] used transposon insertion mutation to establish that if a rich growth medium is provided, only 382 of the protein-coding genes are essential. However, in the organism's natural habitat, the human urogenital tract, probably all 482 are necessary.
The 382 essential genes provide these metabollic pathways:

Next, they proceeded to synthesize the whole genome [Science, 2008, 319, 1215]. The scheme was similar to that for the polio virus:
- Fragments of several hundred bp were obtained commercially
- These were assembled into 5-7 kbp "cassettes" in vitro
  - make "sticky" ends with an exo nuclease
  - anneal next segment
  - ligate
  - amplify by pcr
- 24 to 72 to 144 kbp pieces were made by cloning in E. coli
- E. coli rejected larger pieces, so final assembly was done by cloning in yeast.
- Entire synthetic genome was sequenced to establish validity
To avoid pathogenicity, one gene was replaced with an aminoglycoside resistance gene. "Watermarks" were placed in non-coding regions to identify the construct as synthetic.
Finally, the genome was placed into an M. genitalium from which the "natural" genome had been removed. The bacterium lived happily on a good growth medium.
What's next? Mycoplasma laboratorium?

This page last modified 1:11 PM on Tuesday January 31st, 2012.
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