DNA Packaging in Bacteriophage T4: Organized packing of nucleic acids in biological systems is a fascinating phenomenon. We use bacteriophage T4 as a model system to elucidate the mechanism of DNA packaging in double stranded DNA containing icosahedral viruses. DNA packaging occurs by translocation of DNA into a preformed capsid shell and its packing into a nearly crystalline structure. We employ a combination of molecular genetics, biochemistry, and bioinformatics approaches to elucidate the mechanisms of DNA packaging.
A complex packaging machine assembled at the unique portal vertex of the prohead drives DNA translocation utilizing ATP hydrolysis as the energy source. We have been characterizing the biochemical functions of the principal components of this molecular machine; the motor (gp17), the regulator (gp16), and the portal (gp20).
In collaboration with late Prof. Michael Rossmann's lab at the Purdue University, the X-ray and/or cryo-EM structures of essentially all the packaging machine components including the entire virus capsid have been determined.
In collaboration with Dr. Douglas Smith's lab at University of California San Diego, Dr. Yann Chemla’s lab at University of Illinois, and Dr. Taekjip Ha’s lab at Johns Hopkins University, the dynamics of packaging, mechanisms of DNA translocation, and motor coordination are investigated at single molecule level using dual optical tweezers and Total Internal Reflection Fluorescence microscopy.Bacteriophage T4 for Vaccine Development and Genetic Therapeutics: We have developed novel phage T4 platforms to deliver vaccine antigens and therapeutic genes into human cells. The platform uses the outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) as adapters to decorate phage capsid with pathogen antigens, and CRISPR engineering to incorporate therapeutic genes into phage genome.
A dual vaccine has been designed that confers complete protection against both anthrax and plague in animal models. Recently, a T4-COVID vaccine has been designed that confers full protection against SARS CoV-2 infection in the mouse model.
Our research is funded by the National Science Foundation and the National Institutes of Health.
Fang Q, Tang WC, Tao P, Mahalingam M, Fokine A, Rossmann MG, Rao VB. (2020). Structural morphing in a symmetry-mismatched viral vertex. Nature Communications 2020 Apr 6;11(1):1713. doi: 10.1038/s41467-020-15575-4.
Wieczorek L, Peachman K, Steers N, Schoen J, Rao M, Polonis V, Rao V. (2020) Selection and immune recognition of HIV-1 MPER mimotopes. Virology 550:99-108.
Mo Y, Keller N, delToro D, Ananthaswamy N, Harvey SC, Rao VB, Smith DE. (2020) Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence. Nucleic Acids Res. 48(20):11602-11614.
Kim J, Rao VB, Rao M. (2020) Primary HIV-1 and Infectious Molecular Clones Are Differentially Susceptible to Broadly Neutralizing Antibodies. Vaccines (Basel) 8(4):782.
Islam MZ, Fokine A, Mahalingam M, Zhang Z, Garcia-Doval C, van Raaij MJ, Rossmann MG, Rao VB. (2019) Molecular anatomy of the receptor binding module of a bacteriophage long tail fiber. PLoS Pathogens 2019 Dec 19;15(12):e1008193.
Zhu, J., Tao, P., Mahalingam, M., Sha, J., Kilgore, P., Chopra, A. K. and Rao, V. (2019) A prokaryotic eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells. Science Advances 5, eaax0064.
Ananthaswamy, N., Fang, Q., AlSalmi,W., Jain, S., Chen, Z., Klose, T., Sun, Y., Liu, Y., Mahalingam, M., Chand, S., Tovanabutra, S., Robb, M., Rossmann, M.and Rao, V. B. (2019) A sequestered fusion peptide in the structure of an HIV-1 transmitted founder envelope trimer. Nature Communications 10(1):873.
Ordyan, M., Alam, I., Mahalingam, M., Rao, V. B. and Smith, D. E. (2018) Nucleotide-dependent DNA gripping and an end-clamp mechanism regulate the bacteriophage T4 viral packaging motor. Nature Communications 9(1):5434.
Tao P, Mahalingam M, Zhu J, Moayeri M, Sha J, Lawrence WS, Leppla SH, Chopra AK, Rao VB. (2018) A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague. mBio 9(5). pii: e01926-18.
Tao, P., Zhu, J., Mahalingam, M., Batra, H. and Rao, V.B. (2018) Bacteriophage T4 nanoparticles for vaccine delivery against infectious diseases. Advances in Drug Delivery Reviews pii: S0169-409X(18)30164-9.
Tao, P., Wu, X. and Rao, V.B. (2018) Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9. Science Advances Vol. 4, no. 2, eaar4134.
Lin,S., Alam, T.I., Kottadiel, V., VanGessel, C.J., Tang, W-C., Chemla, Y.R. and Rao,V.B.(2017) Altering the speed of a DNA packaging motor from bacteriophage T4. Nucleic Acids Research 45(19):11437-11448.
Chen Z, Sun L, Zhang Z, Fokine A, Padilla-Sanchez V, Hanein D, Jiang W, Rossman MG, Rao V.B.,(2017) Cyro-EM structure of the bacteriophage T4 isometric head at 3.3 A resolution and its relevance to the assembly of icosahedral viruses. Proc Natl Acad Sci U S A. 2017 Sept 11. pii 201708483.doi:10.1073/pnas.1708483114. [Epub ahead of print].
Tao P., Tang W-C. Zhu J., Rao V.B.(2017) Engineering of Bacteriophage T4Genome Using CRISPR-Cas9. ACS Synth Biol. 2017 July 13 doi:10.1021/accynbio.7b00179 [Epub ahead of print}