# Structure and assembly of dsDNA tailed bacteriophages

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2022 · $459,613

## Abstract

ABSTRACT
Bacteriophages (phages) are viruses that infect bacteria. They have important impacts on human health
through their significant effect on bacterial evolution and pathogenicity as well as from biotechnology
applications. Due to the increasing rise in multi-drug resistance by pathogenic bacteria, new interest has
arisen in developing phages as antibiotics, and they are further being exploited for developing therapeutic
drugs and vaccines. We have long studied the genetics and structure of phage HK97 and others that are
similar to the well-known phage lambda (λ) and belong to the very large family of double-stranded DNA
(dsDNA) tailed phages. The capsid of HK97 encloses the viral genome and assembles from multiple
copies of three subunits – the major capsid protein that includes an assembly (scaffolding) domain, the
protease that removes the scaffold domain after assembly, and a portal protein that assembles as a ring
to initiate capsid assembly, and through which the genome enters and exits the capsid. Interactions
between these subunits are key to understanding assembly and the subsequent maturation that removes
the scaffold domain, enables packaging of the dsDNA, and transforms the capsid into the expanded
mature form. We have developed the tools to explore these processes by mutational and structural
analyses and generated compelling preliminary data on which this proposal is based. An important basis
for the project is structure determination by cryo-electron microscopy (cryoEM) of the first assembled
capsid revealing the unique portal vertex and the organization of the scaffold domain relative to the portal.
Although still at modest resolution, this structure elegantly explains a long-standing puzzle about the
symmetry mismatch between the 12-fold portal ring and the 5 capsomers surrounding it. With this model
we can begin investigating the assembly functions of the subunits through existing and new mutants
probing the portal-scaffold domain interface. We aim to develop this study in three directions. In Aim 1
we will extend the study of the portal organization to higher resolution and explore the consequences of
mutations in the scaffolding domain and the portal, the two components that form the interfaces between
the portal and the major capsid protein. In Aim 2 we will model changes that happen after the initial capsid
assembles as it progresses through proteolysis of the scaffold domain and expansion the mature capsid
conformation. In Aim 3 we will compare assembly of the HK97 capsid with those of several phages with
close structural similarities but which assemble capsids of a different (larger) size from HK97. The
significance of this work is in filling knowledge gaps of how this large family of phages assemble capsids
of varying icosahedral geometry from very similar building blocks, providing a framework for their
development as therapies and use in vaccine research.

## Key facts

- **NIH application ID:** 10382154
- **Project number:** 1R01GM144981-01
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** James F. Conway
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $459,613
- **Award type:** 1
- **Project period:** 2022-09-23 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10382154

## Citation

> US National Institutes of Health, RePORTER application 10382154, Structure and assembly of dsDNA tailed bacteriophages (1R01GM144981-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10382154. Licensed CC0.

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