Mass Spectral Analyses of <i>Salmonella</i> Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids
Aaron Scheuch,
Sara A. M. Moran,
Julia N. Faraone,
Sophia R. Unwin,
Gialinh Vu,
Andrea Denisse Benítez,
Nurul Humaira Mohd Redzuan,
Dana Molleur,
Sammy Pardo,
Susan T. Weintraub,
Julie A. Thomas
Affiliations
Aaron Scheuch
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Sara A. M. Moran
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Julia N. Faraone
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Sophia R. Unwin
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Gialinh Vu
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Andrea Denisse Benítez
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Nurul Humaira Mohd Redzuan
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Dana Molleur
Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Sammy Pardo
Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Susan T. Weintraub
Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Julie A. Thomas
Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY 14620, USA
Salmonella myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the composition of the mature SPN3US head and elucidate how it is modified by proteolysis during assembly, we conducted tandem mass spectrometry analysis of purified virions and tailless heads. Fourteen protease cleavage sites were identified in nine proteins, including eight sites not previously identified in head proteins in vivo. Among these was the maturation cleavage site of gp245 which was identical to the autocleavage site we had previously identified in purified recombinant gp245. Our findings underscore the value of employing multiple mass spectrometry-based experimental strategies as a way to enhance the detection of head protein cleavage sites in tailed phages. In addition, our results have identified a conserved set of head proteins in related giant phages that are similarly cleaved by their respective prohead proteases, suggesting that these proteins have important roles in governing the formation and function of large icosahedral capsids.