T4 Bacteriophage: a fascinating macromolecular machine.
For 45-years, our research has centered on bacteriophage T4, to understand the mechanisms of virus assembly and genome packaging. Our goal is to determine the structures and mechanisms of every component of the T4 virion at near atomic detail.
We use a multi-disciplinary approach, combining biochemistry, molecular genetics, recombinant DNA and CRISPR engineering, bioinformatics, structural biology, and single molecule biophysics, to investigate virus assembly and genome (DNA) packaging. We then translate this wealth of mechanistic knowledge into biomedical applications.
We are establishing new T4-based biotechnology platforms for next generation vaccine designs, targeted in vivo gene therapies, and phage therapies against antibiotic-resistant pathogens.
RESEARCH
DNA packaging motor
Phage T4 packages a large, 56 mm (over 2 inches!) long, linear double-stranded DNA genome into a protein shell by employing a powerful packaging motor. The DNA is folded about 660 times, generating a packing density equivalent to that of DNA in crystal form. It creates a strong internal pressure of about 25-30 atm or 5-6 times the pressure in a champagne bottle.
We continue to refine the detailed T4 packaging mechanism by developing powerful combinatorial mutagenesis and high throughput screening strategies. We analyze mutants by structural and single molecule approaches to tease out the functional signatures of the packaging motor.
Structures
We have determined numerous X-ray and cryo-EM structures of bacteriophage T4 in collaboration with Dr. Michael Rossmann’s laboratory (Purdue University). These include: portal, packaging motor, capsid decoration proteins Hoc and Soc, and entire capsid in different conformations. Recently, in collaboration with Dr. Andrei Fokine (Purdue University), Dr. Lei Sun (Fudan University), and Dr. Qianglin Fang (Sun Yat-sen University), we have determined the atomic structures of neck, tail, baseplate, long tail fiber, and Wac fibers, as well as the entire virion. When combined with genetic and biochemical approaches, we are building the deepest mechanistic understanding of these viral molecular machines.
Single motor dynamics
We analyze the dynamics of the T4 DNA packaging motor. We can measure speed, force, power, pauses and slips, on a single motor at a time, using optical tweezers in collaboration with Dr. Doug Smith (University of California, San Diego) and Dr. Yann Chemla (University of Illinois, Urbana-Champaign). In collaboration with Dr. Taekjip Ha (Harvard University), we image motor dynamics and coordination mechanisms in real-time using single molecule fluorescence.
Our studies led to an electrostatic force-driven motor mechanism. Similar to a 5-cylinder engine, the motor pumps DNA in a piston-like fashion, ~2 base pairs at a time, with a rate of 2,000 bp/sec. Generating a force of about 60 pN and power density twice that of an automobile engine (~5,000 kW/m3), the T4 packaging motor is the fastest and most powerful known to date
Gene therapies
The T4 packaging motor is promiscuous and can fill the head to capacity with any DNA, either short oligonucleotides or its native ~170 kb-long genome (headful packaging). This observation allowed us to establish an assembly line in the test tube to create customized nanoparticles for various genetic diseases.
The large capacity of T4 capsid is engineered by incorporating combinations of therapeutic biomolecules, proteins and DNAs, both inside and outside the capsid. These nanoparticles, referred to as “artificial viral vectors (AVVs)”, deliver payloads into a defective human cell and make precise corrections of the genetic defect.
This modular, plug-and-play, phage T4 assembly platform can be adapted to various genetic diseases and cancers.
Vaccines
We engineer the T4 capsid lattice to display pathogen antigens fused to the non-essential outer capsid proteins Hoc and Soc. With up to 1,125 antigens decorating the capsid, these nanoparticles mimic PAMPs (pathogen associated molecular patterns) exposed on human viral pathogens and stimulate robust immune responses when administered to animals intramuscularly or intranasally.
A series of T4 vaccines have been designed against anthrax, plague, COVID-19, and Flu, which were tested in various animal models including mouse, rat, rabbit, and macaque in collaboration with Dr. Ashok Chopra (UTMB). Vaccines that are currently in development include a ‘universal’ Flu vaccine, and a dengue vaccine.
The T4 vaccines are adjuvant-free, stable at ambient temperature, and confer complete protection against lethal challenges. They can be inexpensively manufactured and distributed, bringing vaccine access to low- and middle-income communities across the globe.
In vivo stem cell therapies
Human hematopoietic stem cell (HSC) therapy has long been investigated for treating genetic diseases and cancers. However, manipulating HSCs ex vivo is challenging and prohibitively expensive. Our ongoing research, funded by NIDA Avant-Garde award, is focused on creating targeted in vivo stem cell therapies. When administered as an i.v. injection, the T4-AVVs would capture HSCs and deliver the payload that carries out genetic repairs to correct the defective gene.
Currently, we are creating AVVs carrying a payload of genome editing molecules that remove the HIV co-receptor gene CCR5 from the HSCs. The genetically modified HSCs then differentiate and repopulate, thus reprogramming the immune reservoir to become HIV resistant resulting in an HIV cure for people living with HIV.
The T4 nano particle is non-infectious, non-toxic, and has no significant pre-existing immunity in humans, and can be inexpensively manufactured in the E. coli bacterium, making the technology accessible to all communities.
COLLABORATORS
A key aspect of our research program is our long-standing collaborations with leaders in
various fields as well as contributions by numerous students and research fellows.
Late Michael Rossmann and Andrei Fokine (Purdue University)
Ashok Chopra and Haitao Hu (University of Texas Medical Branch)
Doug Smith (University of California, San Diego)
Yann Chemla (University of Illinois, Urbana-Champagne)
Taekjip Ha (Harvard University)
Frank Malderelli (NCI Frederick HIV Program)
Our research is funded by the National Science Foundation and National Institutes of Health.
