A Long and Winding (Collaborative) Bacteriophage Road

Sherwood Casjens
Professor Emeritus
School of Biological Science
University of Utah
sherwood.casjens@path.utah.edu

As my time as a graduate student at Stanford University working on bacteriophage lambda waned, I thought I had a fine postdoc in a new research field lined up in England with a very famous Nobel laureate. Then, less than six months before I was scheduled to arrive there I received a letter stating curtly that they were remodeling the lab and would no longer have space for me in my hoped-for time window. I was upset and worried about my future, but luckily I had met Jonathan King (in his CalTech days) at phage meetings. He had recently set up his own lab at MIT, where he was continuing his work at the very cutting edge of macromolecular assembly as exemplified by the tailed bacteriophages. I contacted Jon about a postdoc in his lab, and he accepted me without hesitation – whew, I was very grateful to have dodged that bullet!

Jon’s seminal work at MIT with David Botstein that set up the phage P22 system as one of the premier “model system” phages (Botstein et al., 1973; King et al., 1973) sharpened my interest in phages and helped aim my career down the path that has happily meandered through many aspects of phage biology and evolution (almost always with phage P22 as the study subject) for five decades. In retrospect I have never been sorry for a minute that I missed out on my original postdoc arrangement and am very happy and thankful that Jon took me into his lab. I thoroughly enjoyed my time there, and it allowed me to continue to work with phages, my first and still greatest scientific love.

I arrived at MIT in the fall of 1972 and immediately started work on a phage P22-encoded protein that Jonathan had recently found was required for virion assembly but appeared not to be in the finished virus particles (virions). I was able to show that this protein is indeed required for proper capsid shell assembly and that it functions catalytically with an average molecule participating in about five rounds of virion assembly under our laboratory conditions. Jon coined its name, “scaffolding protein,” which lives on today as one of the important features of the assembly of large virions. I found that there were about 200 molecules of scaffolding protein present in each virus precursor particle (called the prohead or procapsid), while there was less than one molecule per completed virion (King and Casjens, 1974; Casjens and King, 1974). A bit later in my own new lab at the University of Utah, in collaboration with Jon at MIT, we found that scaffolding protein turns off its own synthesis when it is not assembled in proheads (King et al., 1978; Casjens et al., 1985).

Those were heady days at MIT when phages were truly the model system for studying the basic molecular processes of life, and the scientific world looked to phage researchers for insight into molecular biological processes. Our scaffolding protein work was the first to show that a protein could catalyze a non-covalent macromolecular assembly process, and it was very well received. We published a lead article on this in Nature (King and Casjens, 1974), which remains one of my most important papers. The King lab was a small but exciting place to be at the time, with Jon working in the lab, postdoc Yoshiko Kikuchi working out the elegant details of the phage T4 baseplate assembly pathway and Elaine Lenk doing state-of-the-art electron microscopy on phage assembly. Jon was one of the best bench scientists I have known – his pre-experiment organization and sophisticated, precise use of controls remain unmatched in my experience. I learned the use of controls from him so well that due to my continued harping at my students about controls I acquired the nickname “Dr. Controls” in my own lab.

Bill Earnshaw, a new graduate student in the King lab when I was there, and I hit it off well personally and started working together. Bill had the idea to use low-angle x-ray scattering to examine the location of scaffolding protein in proheads, and I had worked out ways to purify large numbers of intact proheads and to specifically remove the scaffolding protein without disrupting the capsid’s protein shell (Casjens and King, 1974). Bill was brave enough to arrange for us to use Steve Harrison’s equipment at Harvard to do this, and we were the first to show unequivocally that scaffolding protein was in the interior of proheads. This had important implications on how scaffolding proteins must function, and we published an important paper describing our work (Earnshaw et al., 1976). I also had good interactions with David Botstein’s research group at MIT and collaborated with Ken Lew there to identify the P22 early gene-encoded proteins in electrophoresis gels (Lew and Casjens, 1975). These positive interactions stimulated my innate urge to collaborate, which has served me very well throughout my career.

Luckily, Jon had visited Albert Einstein Medical School in New York City and discovered that Donald Summers there was in the process of forming a new basic science department at the University of Utah School of Medicine. We (my wife Wai Mun Huang and I) contacted Don and gave job seminars in New York. Scaffolding protein was a big hit, as was Wai Mun’s work on identifying the proteins made by phage T4 early in infection, and we accepted two independent faculty positions in Utah starting in the fall of 1974. To this day we still enjoy living in Utah. Both of us have had very fulfilling careers here, and we enjoy the many nearby outdoor recreation possibilities. Jon was singularly responsible for this, not only by letting me into his lab at short notice and by discovering the Utah job openings, but also for making my research sound more important by kindly giving me full credit for the scaffolding protein work when it was his initial observation that made it all possible and for encouraging me to continue my work on scaffolding protein after I left his lab.

Meanwhile, Jon had been asked to write an article for the Annual Review of Biochemistry while I was at MIT. He generously asked me to co-author it and gave me essentially free rein in the bulk of the writing. This was still a new field, and a general review on the topic had never been written. We decided to make it comprehensive and cover all viruses rather than have a narrow focus on phages, and I spent my last summer there completely submerged in piles journal articles. We finished it as I left for Utah (Casjens and King, 1975). In doing this I learned an incredible amount and inadvertently became one of the (at that time very few) apparent experts on the general field of virus assembly. The fact that this review was especially well received (it was still being cited many years later) gave me my first confidence boost that I could have a positive impact in science beyond the confines of my lab project. I also discovered that I enjoyed trying to bring a lot of disparate facts together and connect them in the context of a review article to form a coherent “big picture”. As a consequence of this great experience with Jon I have enjoyed writing many review articles since then.

Jon continued to impact me in positive but less direct ways long after my MIT days. He continued to have excellent students and postdocs in his lab who worked in phage assembly, all of whom I got to know well at phage and macromolecular assembly conferences. Bill Earnshaw, by then at a postdoc position in England, and I in Utah had noticed independently that there were unifying principles in the studies on phage head assembly that had proliferated to a number of different phages by that time, and we published a review together on this in Cell that also enjoyed an exceptionally long citation life (Earnshaw and Casjens, 1980). In Utah, I was continuing my genetic and molecular biological work on scaffolding protein but soon realized that it would also take more biophysically oriented experiments to truly understand how it works. Peter Prevelige and Carol Teschke, both postdocs from Jon’s lab, had different biophysical expertise skills that were complementary to my largely genetic experience and had established their own labs at U. of Alabama at Birmingham and U. of Connecticut, respectively. Happily, I was able to have productive long-term collaborations with each of them that contributed greatly to our current understanding of the role of scaffolding protein in virus capsid assembly. These joint studies were rewarding and great fun, and, among other things, showed that it is the C-terminal domain of scaffolding protein that binds the inside of the coat protein shell (Parker et al., 1998; Tuma et al., 1998), elucidated the atomic structure of that C-terminal domain (Sun et al., 2000), and determined which amino acids in that structure are important for its coat interaction (Cortines et al., 2011; Padilla-Meier et al., 2012). I also collaborated with Bob Villafane, another student from John’s lab, then on the faculty of Alabama State U., to determine and analyze the genome sequence of the P22 phage relative epsilon34 (Villafane et al., 2008), and also with a student I knew at MIT in the Botstein lab, Tony Poteete, then a professor at U. of Massachusetts, to understand the P22 lysis genes (Casjens et al., 1989). These and other collaborations also included work with P22 on DNA packaging (Lander et al., 2006; Němeček et al., 2007), capsid decoration proteins (Parent et al., 2012; Newcomer et al., 2019), the role of the host in phage protein folding (Gordon et al., 1994), the mechanism of DNA injection into the host bacterium (Leavitt et al., 2021) and even phage evolution and natural diversity (Casjens et al., 1992; Casjens, 2003, 2005; Casjens and Thuman-Commike, 2011; Grose and Casjens, 2014; Casjens and Grose, 2016). The assembly and function of large dsDNA viruses like P22 is very complex and has not always been easy to study. As a result, various parts of the process are still not fully understood, and various aspects of scaffolding protein function as well as other aspects of virion assembly and function remain mysterious to this day. But doesn’t any good science pose more new questions than it has answered? All my collaborations greatly expanded my phage horizons and made my career thoroughly satisfying and enjoyable.

Thank you Jonathan!

Botstein, D., Waddell, C.H., and King, J. (1973). Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation. J. Mol. Biol. 80: 669-695.

Casjens, S. (2003). Prophages in sequenced bacterial genomes: what have we learned so far? Molec. Microbiol. 49: 277-300.

Casjens, S. (2005). Comparative genomics and evolution of the tailed-bacteriophages. Curr. Opinion Microbiol. 8:1-8

Casjens, S., Adams, M., Hall, L., and King, J. (1985). Assembly-controlled autogenous modulation of bacteriophage P22 scaffolding protein gene expression. J. Virol. 53:174-179.

Casjens, S., Eppler, K., Parr, R., and Poteete, A. (1989). Nucleotide sequence of the bacteriophage P22 gene 19 to 3 region: identification of a new gene required for lysis. Virology, 171: 588-598.

Casjens, S., Hatfull, G., and Hendrix, R. (1992). Evolution of the dsDNA tailed-bacteriophage genomes. Seminars in Virology 3: 383-397.

Casjens, S., and Grose, J. (2016). Contributions of P2- and P22-like prophages to understanding the enormous diversity and abundance of tailed bacteriophages. Virology 496: 255-276.

Casjens, S., and King, J. (1974). P22 Morphogenesis I: Catalytic scaffolding protein in capsid assembly. J. Supramol. Str. 2:202-224.

Casjens, S., and King, J. (1975). Virus Assembly. Annual Review of Biochemistry 44:555-771.

Casjens, S., and Thuman-Commike, P. (2011). Evolution of mosaic tailed bacteriophage genomes seen through the lens of phage P22 virion assembly. Virology 411: 393-415.

Cortines, J., Weigele, P., Gilcrease, E., Casjens, S., and Teschke, C. (2011). Decoding bacteriophage P22 assembly: Identification of two charged residues in scaffolding protein responsible for coat protein interaction. Virology 421:1-11.

Earnshaw, W., and Casjens, S. (1980). DNA packaging by the double-stranded DNA bacteriophages. Cell 21:319-331.

Earnshaw, W., Casjens, S., and Harrison, S. (1976). Molecular structure of bacteriophage P22 and its precursor structures I: Low angle x-ray diffraction studies. J. Mol. Biol. 104:387-410.

Gordon, C., Sather, S., Casjens, S., and King, J. (1994). Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins. J. Biol. Chem. 269: 27941-27951.

Grose, J. and Casjens, S. (2014). Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae. Virology 468-470: 421-443.

King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119.

King, J., Hall, L., and Casjens, S. (1978). Control of the synthesis of phage P22 scaffolding protein is coupled to capsid assembly. Cell 15:551-560.

King, J., Lenk, E.V., and Botstein, D. (1973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway. J. Mol. Biol. 80: 697-731.

Lander, G., Tang, L., Casjens, S., Gilcrease, E., Prevelige, P., Poliakov, A., Potter, C., Carragher, B., and Johnson, J. (2006). The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science, 312: 1791-1795.

Leavitt, J., Gilcrease, E., Woodbury, B., Teschke, C., and Casjens, S. (2021). Intravirion DNA can access the space occupied by the bacteriophage P22 ejection proteins. Viruses 13:1504.

Lew, K., and Casjens, S. (1975). Genetic control of the phage P22 early proteins. Virology 68:525-533.

Němeček, D., Gilcrease, E., Kang, S., Prevelige, P. Jr., Casjens, S., and Thomas, G. Jr. (2007). Subunit conformations and assembly states of a DNA translocating motor: The terminase of bacteriophage P22. J. Mol. Biol 374: 817-836.

Newcomer, R., Schrad, J., Gilcrease, E., Casjens, S., Feig, M., Teschke, C., Alexandrescu, A., and Parent, K. (2019). The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy. eLife 8:e45345

Padilla-Meier, G., Gilcrease, E., Weigele, P. Cortines, J., Siegel, M., Leavitt, J., Teschke, C., and Casjens, S. (2012). Unraveling the role of the C-terminal helix-turn-helix of the coat-binding domain of bacteriophage P22 scaffolding protein. J. Biol. Chem. 287:33766-33780.

Parent, K., Deedas, C., Egelman, E., Casjens, S., Baker, T., and Teschke, C. (2012). Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles. Biomaterials 33: 5628-5637.

Parker, M., Casjens, S., and Prevelige, P., Jr. (1998). Functional domains of bacteriophage P22 scaffolding protein revealed by deletion mutagenesis. J. Mol. Biol. 281: 69-79.

Sun. Y., Parker, M., Weigele, P., Casjens, S., Prevelige, P. and Krishna, N. R. (2000). NMR solution structure of the coat protein-binding domain of bacteriophage P22 scaffolding protein.. J. Mol. Biol. 297: 1195-1202.

Tuma, R., Parker, M., Weigele, P., Sampson, L., Sun, Y., Krishna, R., Casjens, S., Thomas, G., and Prevelige, P. (1998). A helical coat protein recognition domain in the P22 scaffolding protein. J. Mol. Biol. 281: 95-106.

Villafane, R., Zayas, M., Gilcrease, E., Kropinski, A., and Casjens, S. (2008). Genomic analysis of bacteriophage e³⁴ of Salmonella enterica serovar Anatum (15+). BMC Microbiology 8: e227