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About viruses and viral disease

bacteriophage

Viruses go green

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Visualization of M13 virus (orange strand to the right) using the surface proteins (purple strands) to grab the carbon nanotubes and put them in order. Image credit: Matt Klug, Biomolecular Materials Group, MIT.

This article was written for extra credit by a student in my recently concluded virology course.

by Ian Blubaugh

A team at MIT has been able to use a genetically engineered virus to help build carbon nanotubes, microscopic cylinders that are integral in constructing high-efficiency solar cells. These nanotubes had proven difficult to construct due to their small size, and traditional techniques were doing a poor job. However, a virus that was custom made for the job was able to build the nanotubes relatively easy and with a significantly higher level of efficiency and is a promising new technology for the environmental industry.

Angela Belcher, a researcher of Biological Engineering at MIT, believes that viruses can help us build a smarter and better future. She was inspired by the discovery of an abalone shell on the beach one day. She admired how such a complex and beautiful structure could be fashioned out of millions of interactions at the nano-level, all done at standard temperature and pressure without the use of any toxins or harmful chemicals, and wondered why the same biological processes could not be used to build any number of complex materials.

Her team began to focus on the production of solar cells, and particularly on the construction of single-walled carbon nanotubes (SWNT), which is a microscopic component in a solar cell that can provide a high-level of electron diffusion without impacting any other design parameters. However the construction of SWNTs has been limited by the difficulty involved in making such a tiny component and in preventing the tubes from ‘bundling’ together. Previous chemical methods have lead to deterioration and caused other negative effects and were unable to show any increase in photocell efficiency.

The research team found that a virus would be the perfect vehicle for overcoming these obstacles. They used the virus M13 as a facilitator to ‘build’ the SWNT pathway. M13 is a very small ssDNA bacteriophage that is relatively simple to genetically engineer, and by making a recombinant M13 that would bind the SWNTs, they developed a nano-sized hardworking middleman who would bind the SWNTs and arrange them on the surface, but also keep them spaced out enough so that they won’t bundle with each other.

Viruses can be a powerful tool in biological engineering because of their incredible ability to replicate, in the proper conditions, quickly and efficiently. Viruses such as M13 are also relatively easy to genetically engineer. The team was able to utilize the M13 virus by genetically engineering, and then selecting for, a virus that expressed the particular peptide chains on the surface that would complement the SWNTs and bind them to the surface. In this way, each virus would grab about 10-12 SWNTs and hold them on the surface. The virus was also modified to express titanium dioxide (TiO2) on the surface which allowed the SWNTs and the TiO2 to come into close contact with each other and fuse. The virus’s role in fusing the SWNTs and the TiO2 is considered to be the big reason for the gain in efficiency. Prior processes had been unable to find a way to develop a strong contact between the two, so the viral role was critical.

For more information on the experiment, check out the MIT article. For a broader talk about the work, listen to Dr. Belcher speak at TED about the powerful new ways that viruses can be used to make cleaner, better materials.

Dang X, Yi H, Ham MH, Qi J, Yun DS, Ladewski R, Strano MS, Hammond PT, & Belcher AM (2011). Virus-templated self-assembled single-walled carbon nanotubes for highly efficient electron collection in photovoltaic devices. Nature nanotechnology, 6 (6), 377-84 PMID: 21516089

TWiV 135: Live in the Big Easy

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ASM GM New OrleansHosts: Vincent Racaniello, Roger Hendrix, Rachel Katzenellenbogen, and Harmit Malik

Vincent and guests Rachel Katzenellenbogen, Roger Hendrix, and Harmit Malik recorded TWiV #135 live at the 2011 ASM General Meeting in New Orleans, where they discussed transformation and oncogenesis by human papillomaviruses, the amazing collection of bacteriophages on the planet, and the evolution of genetic conflict between virus and host.

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Weekly Science Picks

Roger – Atomic structure of adenovirus by cry0-EM (Science)
Harmit – Syncytin knockout mice show role for endogenous retroviral gene (PNAS)
 Vincent – Free science, one paper at a time by David Dobbs

Listener Pick of the Week

Mark  – Shot by Shot

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TWiM 6: Antibacterial therapy with bacteriophage: Reality or fiction?

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bacteriophage modelHosts: Vincent Racaniello, Cliff Mintz, Michael Schmidt, and Elio Schaecter

On episode #6 of the podcast This Week in Microbiology, Vincent, Cliff, Michael and Elio review the use of bacteriophages to manage infections, and the presence of antibiotic resistance genes in bacteriophages from urban sewage and river water.

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The model of bacteriophage T4 shown in the photo is described here.

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Rich Condit reminisces

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infectious polio rnaOn my recent trip to record TWiV #111 at Florida Gulf Coast University, I visited Rich Condit in Gainesville. There he told me a story about how the bacteriophage T7 polymerase/promoter system was developed. It’s an interesting tale that demonstrates how important scientific advances often have convoluted roots. You can watch the video below or download a high-definition (720p) version (585 MB .mov).

The combination of bacteriophage T7 RNA polymerase and its cognate promoter sequence allows the production of specific RNAs either in vitro or in cells. In many laboratories this system is used to synthesize infectious viral RNA from cloned DNAs. In the example shown, a DNA copy of of the poliovirus RNA genome has been cloned into a bacterial plasmid. The promoter sequence for T7 RNA polymerase is inserted at the 5′-end of the viral sequence (not shown). When placed within any double-stranded DNA, this 19 nucleotide promoter sequence (TAATAGGACTCACTATAGG) will lead to the production of RNA. The plasmid is incubated with purified T7 RNA polymerase (commercially available) and ATP, GTP, UTP and CTP, and the viral RNAs that are produced are then transfected into cells. A viral replication cycle begins, resulting in the production of infectious virus particles.

As described by Rich in the video below, this expression system was conceived by Ed Niles and brought to practice by Bernard Moss. The first paper published described the insertion of the gene encoding T7 RNA polymerase into the genome of vaccinia virus. When cells are infected with the recombinant virus, they produce T7 RNA polymerase. To express a protein using this system, a gene is cloned into a plasmid next to the T7 RNA polymerase promoter. When this plasmid is introduced into cells producing T7 RNA polymerase, RNAs are made which are then translated into protein.

This method was subsequently modified by Wimmer and colleagues to synthesize infectious poliovirus RNA in vitro.

Werf, S. (1986). Synthesis of Infectious Poliovirus RNA by Purified T7 RNA Polymerase Proceedings of the National Academy of Sciences, 83 (8), 2330-2334 DOI: 10.1073/pnas.83.8.2330

Fuerst, T. (1986). Eukaryotic Transient-Expression System Based on Recombinant Vaccinia Virus that Synthesizes Bacteriophage T7 RNA Polymerase Proceedings of the National Academy of Sciences, 83 (21), 8122-8126 DOI: 10.1073/pnas.83.21.8122

Bats harbor many viral sequences

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How large is the zoonotic pool – all the animal viruses that could one day infect humans? Assuming that there are 50,000 vertebrates on earth, each with 20 viruses, the number is one million – probably a vast underestimate. Determining just how many viruses exist in a variety of animal species is technically feasible, limited only by the number of hosts that can be sampled. A study of the virome of several North America bats reveals that these animals – which constitute 25% of all the known mammalian species – harbor a very large collection of viral sequences.

Advances in nucleotide sequencing technologies (deep sequencing) have made it possible in recent years to study the virome – the genomes of all viruses in a host – in human blood, diarrhea, and respiratory secretions; grapevines, and feces of horses and bats. The latter mammals are a particularly important subject because it is known that they harbor the predecessors of several important human viruses, including SARS coronavirus, ebolavirus, marburgvirus, Nipah, Hendra, and rabies viruses. Since there are about 1200 known species of bats, the potential for future human zoonoses is significant.

A multicenter group comprising virologists* and chiropterists (scientists who study bats) has examined the virome of three North American bat species: big brown bats (Eptesicus fuscus), tri-colored bats (Perimyotis subflavus), and little brown myotis (Myotis lucifugus). Deep sequencing was used to analyze fecal and oral samples from 41 bats captured on one night in Western Maryland. The results provide a comprehensive glimpse of the bat virome.

The 576,624 sequence ‘reads’ (a read is the result of a single sequence reaction, in this study ~250 nucleotides) on six pools of fecal samples revealed an amazing diversity of viral sequences (figure), with representatives of human, other mammals, insect, bacteriophage, fish, shrimp, protist, reptile, plant, avian, fungi, algae, and marine viruses. Among the interesting findings are sequences of three novel coronaviruses from big brown bats. Many coronaviruses have previously been found in bats; the results of this study provide more evidence that these mammals are likely to be sources of future human infections.

Novel viruses of plants and insects were also identified in fecal samples from all bats. This observation can be explained by the bats’ prodigious appetite for insects, which harbor both insect or plant viruses (the latter transmitted to plants by insect vectors). It seems unlikely that these viruses replicate in bats, but pass through the gastrointestinal tract into the feces.

Perhaps not surprisingly (given the diversity of bacteria that colonize mammalian intestinal tracts), sequences of novel bacteriophages were also identified in fecal samples. One appears to have high sequence identity with a bacteriophage that infects the plague bacterium Yersina pestis. Curiously, such bacteria are not known to colonies animals in the northeastern United States. A second bacteriophage infects strains of E. coli associated with human illnesses. These findings suggest that bats could be involved in the spread of human bacterial pathogens.

The main viral sequences identified in pooled oral samples were of a novel cytomegalovirus. These viruses appear to be common in bats, and have been been detected in many previous studies.

One question that arises from these findings is whether bats are unique in harboring a large collection of diverse viruses. The answer to this question awaits studies of the viromes of other wild animals.

Viral discovery by massive sequencing will no doubt identify many new viruses in a wide range of species. However, this technology cannot answer some of the more intriguing biological questions, such as which hosts support viral replication, and whether it is associated with disease. Answers to these questions will require construction of complete DNA copies of viral genomes and recovery of infectious viruses by transfection of cells in culture.

The title of this post is ‘Bats harbor many viral sequences’, not ‘many viruses’. That’s because no infectious viruses were identified – only parts of their genomes. If you wish to conclude that a certain virus infects bats, you must either isolate the virus in cell culture, or show that the entire viral genome is present in tissues or fluids.

*including Eric F. Donaldson and Matt Frieman, who spoke about this work on TWiV 90 and 65.

Donaldson EF, Haskew AN, Gates JE, Huynh J, Moore CJ, & Frieman MB (2010). Metagenomic Analysis of the Virome of three North American Bat Species: Viral Diversity Between Different Bat Species that Share a Common Habitat. Journal of virology PMID: 20926577

A new type of enveloped virus?

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All known virus particles can be placed into one of two general categories: enveloped or non-enveloped. Viruses that fall into the former category are characterized by a lipid membrane derived from the host cell, and one or more nuclecapsid proteins that interact with the viral genome. A virus that infects an archaeal host may constitute a new category of enveloped viruses. It comprises a membrane vesicle that encloses a circular ssDNA genome which is devoid of nucleic acid-binding nucleoproteins.

Examples of enveloped virions that contain nucleoproteins are shown in the figure below. These include influenza virus (left), a simple retrovirus (center), and a togavirus (right).

The influenza virion contains segments of viral RNA bound to four different proteins. Retroviral RNA is bound to a nucleocapsid protein which in turn is enclosed in a capsid, while togavirus RNA is located within an icosahedral shell.

Until recently, it was believed that the genome of all other known enveloped DNA and RNA viruses is always associated with one or more viral proteins. This belief may be changed by the isolation, from a solar saltern in Trapani, Italy, of a virus that infects the archaeal species Halorubrum. Salterns are multi-pond systems in which sea water is evaporated to produce salt. In such hypersaline envrionments, Archaea predominate, and about 20 archaeal viruses have been isolated from these locations.

The virus isolated from the Italian saltern is called Halorubrum pleomorphic virus-1, or HRPV-1. Biochemical analyses of the virion show that it is composed of lipids and two structural proteins, VP3 and VP5. The genome is a circular ssDNA about 7 kb in length with nine open reading frames. The virion architecture is unique: it is composed of a flexible membrane (hence the designation pleomorphic) that contains external spikes of the VP4 protein, and is lined on the interior with VP3. The viral DNA is apparently not bound to any proteins in the virions.

At the upper left is my depiction of the appearance of HRPV-1. The diagram was produced by deleting the internal proteins and nucleic acid of a simple retrovirus and replacing these with a ssDNA genome. The HRPV-1 VP4 spikes and the internal VP3 proteins are present, but no proteins are bound to the viral genome. Whether or not the VP4 spikes are oligomeric as shown is unknown.

Most enveloped viruses acquire their lipid membrane by budding from the host cell, and a similar mechanism could account for the formation of HRPV-1 virions. In the absence of a nucleoprotein, it is not clear how the viral genome would be specifically incorporated into the budding envelope. Another condundrum is how the virions would pass through the proteinaceous layer that covers the archaeal host cell.

Whether HRPV-1 is representative of a new kind of virus lacking nucleocapsid protein will be revealed by the study of other pleomorphic enveloped viruses. Candidates include bacterial viruses that infect mycoplasmas, and another pleomorphic haloarchaeal virus isolated from a different Italian saltern, Haloarcula hispanica pleomorphic virus 1.

Pietila, M., Laurinavicius, S., Sund, J., Roine, E., & Bamford, D. (2009). The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes Journal of Virology, 84 (2), 788-798 DOI: 10.1128/JVI.01347-09