Image from Victor Padilla-Sanchez, PhD/Creative Commons Attribution 4.0 International
With SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus), the cause of the current pandemic named Covid-19 (for Corona Virus Disease 2019), I have realized that my life story could be told around viruses. Viruses have played a role in my life at every stage of my life and not just in getting sick from them. Here is the beginning of what I hope to be a series of blog posts that chronicle this virus infected life now in its sixth decade.
But first of all, what is a virus? Essentially a virus is small particle that contains a segment of genetic material, either DNA or RNA, surrounded by a protein coat, sometimes with lipid components like in cell membranes. Viruses are in tenths or hundreds of a micron (1/1000 of a millimeter) in size, smaller than typical bacteria cells but larger than large biological macromolecules like proteins. It is not a cell and it does not carry out metabolism, thus it is not consider to be a living thing. However, it reproduces, evolves, and is composed of biological molecules. Its genetic material has genes that code for the coat proteins and proteins (mostly enzymes) that enable the virus to use host cell functions (DNA replication, RNA transcription, protein synthesis, etc.) to make and release new virus particles. The virus binds to the host cell and injects its genetic material or gets absorbed into the cell by endocytosis. Once progeny virus particles are produced, the cell either breaks open and releases many newly synthesized virus particles or mature virus particles are released by normal bulk transport methods such as exocytosis. The figure above show an schematic of one of my favorite viruses (because of its spaceship appearance) bacteriophage T4. Literally, bacteriophage means bacteria eater–it is a virus that infects bacteria. Here you see the virus particle landing on a bacterial surface at a particular protein binding site. It then injects its DNA into the cell. The virus has a capsid, the protein coated head which contains the DNA and a tail with a sheath, tail fibers, and a baseplate that docks to the cell surface.
Like most Americans I was vaccinated as an infant against diphtheria, tetanus, and pertussis, the DPT shot, smallpox, and polio. Diphtheria, tetanus, and pertussis are caused by bacteria, but smallpox and polio are viral infection. Because of vaccinations these diseases are no longer the problem they once were. Smallpox has been eradicated from the world, the last case having occurred in the 1970s. Smallpox vaccines have been discontinued. There are two stocks of the virus in the world, one in the US and one in Russia.
Having been born into the world too early to get the vaccines available today, I gained immunity to mumps, measles, rubella, and chicken pox the painful way, by experiencing these childhood diseases. Now vaccines are available for these formerly common childhood diseases. So far my experience with viruses is fairly typical of an American my age.
The twist that got me thinking about this story is my experiences as a molecular biology undergraduate at Purdue University and as a graduate student in molecular biology at the University of Oregon. At Purdue I landed an undergraduate research position in the protein crystallography lab of Professor Patrick Argos. In the late 1970s Pat was part of a group of structural biologists that also included Michael Rossmann and Jack Johnson. The mainstay of this group was Michael Rossmann–Pat ended up at the European Molecular Biology Laboratory (EMBL) and Jack at Scripps Institute. Protein crystallography and structural biology is the science of determining the three-dimensional molecular structure of large molecules like proteins made of tens of thousands of atoms. The technique is to grow crystals of these molecules, to expose these crystals to a beam of x-rays, to collect the scattered x-rays, then to reassemble the scattered light using computers to get the molecular structure. The Rossmann, Johnson, Argos lab were in a race with Steve Harrison at Harvard to get the first 3D structure of a spherical plant virus. The Purdue group worked on southern bean mosaic virus (SBMV) and the Harvard group worked on tomato bushy stunt virus (TBSV). Sadly for us, the Purdue group came in second place in that race, but there are some interesting stories to tell about that time.
My role as an undergraduate was rather minor. We had to grow these virus in one of the university greenhouses. One of my jobs was to smash up infected plant leaves mixed with some abrasive material and to rub them on the leaves of young plants. These plants would become infected with the virus and later harvested. From there our biochemistry lab technician would purify the virus from the plant material and grow crystals. Another task I had during those years was to use a newfangled computer graphics system called Molecular Modeling System-X (MMS-X) to build the molecular structure of one of the subunits of the virus coat protein into the electron density map (the result of final calculations in the protein crystallography experiment). The basic structure had already been determined and published, this was sort of mop up work, but an amazing privilege for an undergraduate. I was a newbie and it showed. I built a very bad structure–so bad that Dr. Rossmann gave me a B instead the normal A for such research courses. I’ll detail that story later. Michael Rossmann remained active as a structural biologist until his death in 2019. His group had determined the structure of the virus that causes the common cold and most recently the structure of the zika virus.
As I result of that experience at Purdue, I became a protein crystallographer and went to the University of Oregon to work in the lab of Brian Matthews and get my Ph.D. There I became involved in the T4 lysozyme project. T4 is a bacteriophage, a virus that infects E. coli bacteria. T4 lysozyme is a protein (an enzyme) that breaks down the bacterial cell wall so that newly made virus particles are released from the infected cell to go find other cells to infect. T4 lysozyme had been used in the research of molecular geneticist George Streisinger to confirm in vivo the genetic code, that relationship between a protein sequence and the coding DNA sequence. In the course of the Streisinger research hundreds of mutants (proteins with alternate sequences) had been generated. Brian Matthews chose to work on a career spanning research program of relating the amino acid sequence of a protein to its structure, stability, and folding. By the end of my graduate career we were using newly developed recombinant DNA techniques to grow mutant proteins, but at the beginning I was using the techniques of bacterial virus genetics to make new mutants. To obtain enough T4 lysozyme to make crystals, we would make about 10 gallons of bacterial culture and then infect it with the virus. In the end all the bacteria would be dead and we would have a solution of viruses and the protein, T4 lysozyme, which could be purified, concentrated, and crystallized.
The bacteriophage lambda was also a subject of research at U of O. Whereas bacteriophage T4 is a lytic virus, lambda is a temperate or lysogenic virus. A lytic virus infects cells, makes new virus parts and DNA, and then breaks open the cell releasing 100-150 new virus particles. Phage lambda can do the same, but it also has a mode where it incorporates into the bacterial DNA, and its DNA replicates along with the bacterial DNA. Only under certain stressful conditions does it go back to a lytic phase. Phage lambda was used in research to study aspects of molecular genetics, and it was used in teaching labs for biology and genetics courses. I took some of those courses and was teaching assistant in others.
I also encountered the E. coli virus M13. This virus was used in DNA sequencing experiments which I used in my research to study T4 lysozyme mutants. You can readily isolate M13 double stranded DNA into which you could insert a piece of foreign DNA using recombinant DNA techniques. But you could also isolate M13 single stranded DNA from which you could do DNA sequencing experiments. Because of the tubular structure of this virus you could insert segments of DNA and the virus would just get longer. With a minor extension of DNA sequencing techniques you could introduce specific mutations into the gene via a technique called site-directed mutagenesis.
The Matthews lab had just begun using the these molecular biology techniques when I first arrived there in 1980. I spent many hours learning these methods and then using them in my research.
My experience with viruses beyond these early academic experiences are like most other people. I have personally experienced common cold, influenza, noravirus infections. One of our daughters had to be hospitalized with a rotavirus infection. We hear of adenoviruses as ways of doing gene therapy. Society over the past few decades has gone through AIDS-HIV, hepatitis, Ebola, SARS, MERS, swine flu (H1N1), Now we are experiencing the Corona virus that causes CoViD-19 which is affecting our lives today in dramatic ways.
Terry M. Gray
April 6, 2020