The main emphasis of this grant is to understand how a protein nanomachine is assembled from its parts, how it functions once assembled, and how the mechanisms of assembly support the nanomachine's biological functions. To address these questions we will use the capsid of bacteriophage HK97 and the tail of bacteriophage??? Both of these offer experimental access to centrally important aspects of biological assembly: The Delta domain of the HK97 capsid protein is an assembly chaperone that mediates assembly of capsomers into capsid and also binds the protease and brings it into the inside of the capsid. It also has a role in getting the portal into position and probably has a crucial role in nucleating capsid assembly as a result. For the tails our recent discovery of the tail assembly chaperones and the way they interact with the tape measure protein and with the tail tube protein illuminates the main features of tail assembly in a way that was not previously clear. Both of our model systems also provide examples of how size can be determined in biological assembly. HK97 assembles T=7 capsids but D3, which has a very similar capsid protein sequence, assembles a T=9. The tail assembles to a length that we showed years ago is determined by the tape measure protein. Our recent result with the assembly chaperones clarifies how the length information in the tape measure protein is translated into tail length. Our proposed experiments with the HK97 heads will include generating mutants of the capsid protein chosen to alter the process of capsid maturation. These will be evaluated by their biochemical and structural phenotypes and interpreted in the context of the wealth of structural and structural/dynamic data available from earlier studies. We will pay particular attention to the Delta domain of the capsid protein, which has crucial roles in several aspects of capsid protein function. For tails we will concentrate initially on investigating the newly identified assembly intermediate, a complex of the tape measure protein and the two assembly chaperones, for which we will examine the structure and assembly activities. We will begin experiments to determine the structure and function of the tail tip, including the minor proteins and the newly discovered iron-sulfur cluster We propose to characterize two examples of non-canonical translation of a head protein (TerL), and to carry out a basic characterization of a newly discovered bacteriophage of a previously unknown type.

Public Health Relevance

In this project we will investigate how viruses get assembled from their parts and we will carry out the investigation with Bacteriophage viruses for two reasons. First, they are extremely experimentally tractable, and solid answers to experimental questions can be obtained. Second, bacteriophages are related to viruses that cause human disease, particularly herpes virus, so what we learn about how bacteriophages function will be informative about how herpes viruses and other viruses of eukaryotic hosts function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Sakalian, Michael
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University of Pittsburgh
Schools of Arts and Sciences
United States
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Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W et al. (2017) Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly. J Mol Biol 429:2474-2489
Tso, DanJu; Peebles, Craig L; Maurer, Joshua B et al. (2017) On the catalytic mechanism of bacteriophage HK97 capsid crosslinking. Virology 506:84-91
Hua, Jianfei; Huet, Alexis; Lopez, Carlos A et al. (2017) Capsids and Genomes of Jumbo-Sized Bacteriophages Reveal the Evolutionary Reach of the HK97 Fold. MBio 8:
Huet, Alexis; Duda, Robert L; Hendrix, Roger W et al. (2016) Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex. J Mol Biol 428:165-181
Casjens, Sherwood R; Hendrix, Roger W (2015) Bacteriophage lambda: Early pioneer and still relevant. Virology 479-480:310-30
Cardone, Giovanni; Duda, Robert L; Cheng, Naiqian et al. (2014) Metastable intermediates as stepping stones on the maturation pathways of viral capsids. MBio 5:e02067
Xu, Jun; Hendrix, Roger W; Duda, Robert L (2014) Chaperone-protein interactions that mediate assembly of the bacteriophage lambda tail to the correct length. J Mol Biol 426:1004-18
Pope, Welkin H; Jacobs-Sera, Deborah; Russell, Daniel A et al. (2014) Genomics and proteomics of mycobacteriophage patience, an accidental tourist in the Mycobacterium neighborhood. MBio 5:e02145
Tso, Dan-ju; Hendrix, Roger W; Duda, Robert L (2014) Transient contacts on the exterior of the HK97 procapsid that are essential for capsid assembly. J Mol Biol 426:2112-29
Oh, Bonnie; Moyer, Crystal L; Hendrix, Roger W et al. (2014) The delta domain of the HK97 major capsid protein is essential for assembly. Virology 456-457:171-8

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