1. 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.
Phage DNA packaging is also an excellent model system to understand the mechanisms of DNA condensation in biological systems and ATP energy transduction in molecular motors.
We employ a combination of molecular genetics, recombinant DNA,biochemical 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. The principal components of the pump, the gene products 16 and 17, have been cloned, overexpressed, and purified. We have developed powerful combinatorial mutagenesis approaches to map the DNA translocating ATPase in gp17. Biochemical characterization of the gp16-gp17 complex and molecular understanding of the linkage between ATP hydrolysis and DNA movement are the principal projects in the lab.
In collaboration with Dr. Michael Rossmann's lab at the Purdue University, the x-ray structures of the ATPase and nuclease domains as well as the full-length gp17 motor protein have been solved recently. The cryo-electron microscopy structure of the DNA packaging motor has been determined.
In collaboration with Dr. Douglas Smith's lab at the University of California, San Francisco, the dynamics of packaging at the single molecule level are being investigated.
2. Bacteriophage T4 for Vaccine Development: We have developed novel strategies to use phage T4 for display of multiple vaccine epitopes on T4 capsid surface. The DNA fragments corresponding to the vaccine epitopes are fused in-frame to the two non-essential outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein). The fusion proteins, under appropriate genetic backgrounds, are assembled onto the Hoc- Soc- capsids. These recombinant phage displaying foreign epitopes are used as potential vaccines for elicitation of protective immune responses. This system is currently being developed to construct multicomponent vaccines against anthrax, plague and HIV.
3. Structural Analysis of Phage T4 Assembly: In collaboration with Dr. Michael Rossmann's lab at the Purdue University, West Lafayette, IN, we have been working on the structural analysis of bacteriophage T4, capsid assembly, and the DNA packaging motor.
The cryo-EM reconstructions of prolate phage T4 with anthrax toxin heptamers displays on the capsid surface have been recently generated. This and other ongoing reconstructions will have implications for vaccine development.
The assembly of "neck" to the capsid in order to seal the packaged DNA and attach tail is being investigated by cryoEM and X-ray structures of the complexes.
Our research is funded by the National Science Foundation and the National Institutes of Health.
Tao,P., Wu, X. and Rao, V.B.(2018) Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9 Science Advances (in press).
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}
Chand S., Messina E.L., AlSalmi W., Ananthaswamy N., Gao G., Uritskiy G., Padilla-Sanchez V., Mahalingam M., Peachman K.K., Robb M.L., Rao M., Rao V.B. (2017) Glycosylation and oligomeric state of envelope protein might influence HIV-1 virion capture by α4β7 integrin. Virology. pii:S0042-6822(17)30164-2. doi: 10.1016/j.virol.2017.05.016
Kim J., Jobe O., Peachman K.K., Michael N.L., Robb M.L., Rao M., Rao V.B.,(2017) Quantitive analyses reveal distinct sensitivities of the capture of HIV-1 primary viruses and psedoviruses to broadly neutralizing antibodies. Highlighted in Virology Virology. pii:S0042-6822(17)30165-4.doi: 10.1016/j.virol.2017.05.015.
Tao P., Li Q., Shivachandra S.B., Rao V.B., (2017) Bacteriophage T4 as a Nanoparticle Platform to Display and Deliver Pathogen Antigens: Construction of an Effective Anthrax Vaccine Methods Mol Biol. 1581:255-267. doi:10.107/978-1-4939-6869-5_15.
Tao, P., Mahalingam, M., Zhu, J., Moayeri, M., Kirtley, M., Fitts, E., Andersson, J., Lawrence, W., Leppla, S., Chopra, A. and Rao, V.B. (2017) A Bivalent Anthrax-Plague Vaccine That Can Protect Against Two Tier-1 Bioterror Pathogens, Bacillus anthracis and Yersinia pestis. Front. Immunol. doi: 10.3389/fimmu.2017.00687.
Tao, P., Mahalingam, M., Rao, V.B. (2016) Highly Effective Soluble and Bacteriophage T4 Nanoparticle Plague Vaccines Against Yersinia pestis. Methods Mol. Biol. 1403:499-518. doi: 10.1007/978-1-4939-3387-7_28.
Gao, S., Zhang, L., Rao, V.B. (2016) Exclusion of small terminase mediated DNA threading models for genome packaging in bacteriophage T4. Nucleic Acids Res. 44(9):4425-4439.
Rao, V.B. and Feiss, M. (2015) Mechanisms of DNA Packaging in Large Double Stranded DNA Viruses. Ann. Rev. Virology 2(1):351-378.
Alsalmi, W., Mahalingam, M., Ananthaswamy, N., Hamlin, C., Flores, D., Gao, G., Rao, V.B. (2015) A New Approach to Produce HIV-1 Envelope Trimers: Both cleavage and proper glycosylation are essential to generate authentic trimers. J. Biol. Chem. 290(32):19780-95.
Sun, L., Zhang, X., Gao, S., Rao, P.A., Padilla-Sanchez V., Chen, Z., Sun, S., Xiang, Y., Subramaniam S., Rao, V.B. & Rossmann, M.G. (2015) Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution. Nature Communications 6:7548.
Kondabagil, K., Dai, L., Vafabakhsh, R., Ha, T.J., Draper, B. and Rao, V.B. (2014) Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function. Virology 468-470:660-668.
Vafabakhsh, R., Kondabagil, K., Earnest, T. M., Lee, K. S., Zhang, Z., Dai, L., Dahmen, K. A., Rao, V. B.and Ha, T. (2014) Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4. Proc. Natl. Acad. Sci. USA 111(42):15096-15101.
Migliori, A. D., Kellera, N., Alam, T. I., Mahalingam, M., Rao, V. B., Arya, G., and Smith, D. E. (2014) Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor. Nature Communications 5: 4173.
Padilla-Sanchez, V., Gao, S., Kim, H. R., Kihara, D., Sun, L., Rossmann, M. G. and Rao, V. B. (2013) Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine. J. Mol. Biol. 426:1019-1038.
Rao, M., Peachman, K. K., Kim, J., Gao, G., Alving, C. A., Michael, N. L., Rao, V. B. HIV-1 Variable Loop 2 and its Importance in HIV-1 Infection and Vaccine Development. (2013) Curr. HIV Research 11:427-438.
Tao, P., Mahalingham, M., Kirtley, M., van Lier, C. J., Sha, J., Yeager, L. A., Chopra, A. K. and Rao, V. B.(2013). Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersenia pestis as next generation plague vaccines. PLOS Pathogens 9(7): e1003495.
Tao, P., Mahalingham, M., Marasa, B., Chopra, A. and Rao, V. B. (2013). In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine. Proc. Natl. Acad. Sci. USA110:5846-5851.
Fokine, A., Zhang, Z., Kanamaru, S., Bowman, V. D., Aksyuk, A. A., Arisaka, F., Rao, V. B. and Rossmann, M. G. (2013). The molecular architecture of the bacteriophage T4 neck. J. Mol. Biol. 425:1731-1744.
Gao, G., Wieczorek, L., Peachman, K. K., Polonis, V. R., Alving, C. R., Rao, M., Rao, V. B. (2013)Designing a soluble near full-length HIV-1 GP41 trimer. J. Biol. Chem. 288:234-46.
Kottadiel, V. I., Rao, V. B., Chemla, Y. R. (2012) The dynamic pause-unpackaging state, an off-translocation recovery state of a DNA packaging motor from bacteriophage T4. Proc. Natl. Acad. Sci. USA109:20000-20005.
Black, L.W. and Rao, V.B. (2012) Structure, Assembly, and DNA packaging of bacteriophage T4 head Adv. Virus Res: 119-153.
Hegde, S., Padilla-Sanchez, V., Draper, B., and Rao, V. B. (2012) Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicates a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation. J. Virol. 86: 4046-57.
Black, L.W. and Rao, V.B. (2012) Structure, Assembly, and DNA packaging of bacteriophage T4 head. Adv.Virus Res. 82:119-53.
Feiss M, Rao VB. (2012) The bacteriophage DNA packaging machine. Adv. Exp. Med. Biol.726:489-509.
Rossmann MG, Rao VB. (2012) Viruses: sophisticated biological machines. Adv. Exp. Med. Biol. 726:1-3.
Sun S, Gao S, Kondabagil K, Xiang Y, Rossmann MG, Rao VB. (2012) Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages. Proc Natl Acad Sci U S A. 109(3):817-22.
Peachman KK, Li Q, Matyas GR, Shivachandra SB, Lovchik J, Lyons RC, Alving CR, Rao VB, Rao M. (2012). Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores. Clin Vaccine Immunol. 19(1):11-6.
Kondabagil, K., Draper, B., Rao, V.B. (2012). Adenine recognition is a key checkpoint in the energy release mechanism of phage T4 DNA packaging motor. J. Mol. Biol. 415(2):329-42.
Fokine, A., Islam, M.Z., Zhang, Z., Bowman, V.D., Rao, V.B., Rossmann, M.G. (2011). Structure of the Three N-terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-like Bacteriophage. J Virol. 85:8141-8.
Alving, C.R., Rao, M., Peachman, K.K., Asher, L., Rao, V.B. (2011) Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembly nanocarrier formulation. J. Liposome Res. 21(4):279-85.
Ghosh-kumar, M., Alam, T.I., Draper, B., Stack, J. and Rao, V.B. (2011) Regulation by Interdomain Communication of a Headful Packaging Nuclease from Bacteriophage T4. Nucleic Acids Res. 39: 2742-2755.
Zhang, Z., Kottadiel, V., Vafabakhsh, R., Li, Dai, Chemla,Y.R., Ha, T.J. and Rao, V.B. (2011) A promiscuous DNA packaging machine from bacteriophage T4. PLOS Biology 9:e1000592, 1-11.("Research Highlights" Nature Reviews Microbiology, 2011 Vol.9)
Rao, M., Peachman, K., Li, Q., Matyas, G., Shivachandra, S., Borschel, Morthole, V.I., Fernandez-Prada, C. R., Alving, C. and Rao, V.B. (2011) Highly effective generic adjuvant systems for orphan or poverty-related vaccines. Vaccine 29:873-877.
Gao, S. and Rao, V.B. (2011) Specificity of interactions among the DNA packaging machine components of T4 related bacteriophages. J. Biol. Chem. 286:3944-3956.
Rao, V.B. and Black, L.W. (2010) Structure and Assembly of Bacteriophage T4 Head. Virology J. 7:356-367.
Tsay, J.M., Sippy, J., DelToro, D., Andrews, B.T., Draper, B., Rao, V., Catalano, C.E, Feiss, M, Smith, D.E. (2010) Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity. J. Biol. Chem. 285:24282-24289.
Sathaliyawala, T., Islam, M.Z., Li, Q., Fokine, A., Rossmann, M.G., Rao, V.B. (2010). Functional analysis of highly antigenic outer capsid protein, Hoc, a virus decoration protein from T4-like bacteriophages. Mol. Microbiol. 77: 444-55.
Sun, S., Rao, V.B. and Rossmann, M. Genome packaging in viruses. (2010) Curr. Opin. Struct. Biol.20(1):114-120.
Li, Q., Fokine, A., O’Donnell, E., Rao, V. B., Rossmann, M. R. (2010) The Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages. J. Mol. Biol. 395:728-741.
Al-Zahrani, A.S., Kondabagil K., Gao, S., Kelly, N., Ghosh-Kumar, M. and Rao, V. B. (2009) The small terminase, gp16, of bacteriophage T4 is a regulator of the DNA packaging motor. J. Biol. Chem 84:24490- 24500.
Rao, V. B. (2009) A virus DNA gate: zipping and unzipping the packed viral genome. Proc. Natl. Acad. Sci. USA 106: 8403-8404.
Kearney, M., Maldarelli, F., Shao, W., Margolick, J.B., Daar, E.S., Mellors, J.W., Rao, V.B., Coffin, J.M. and Palmer, S. (2009) Human immunodeficiency virus type 1 population genetics and adaptation in newly infected individuals. J. Virology 83:2715-27.
Sun, S, Kondabagil, K., Draper, B., Alam, I.T., Baumann, V., Zhang, Z., Hegde., Fokine, A., Rossmann, M.G., and Rao, V.B. (2008) The atomic structure of bacteriophage T4 DNA packaging motor suggests a mechanism dependent on electrostatic forces. Cell 135:1251-1262.
This work was featured as a "Leading Edge" article; Williams, R.S., Williams, G.J. and Tainer, J.A. Cell 135:1169-71.
Rao, V.B. and Feiss, M. The bacteriophage DNA packaging motor. (2008) Ann. Rev. Genetics 42:642-681.
Alam, I. T. and Rao, V. B. (2008) The ATPase domain of the large terminase protein, gp17, from bacteriophage T4 binds DNA: implications to the DNA packaging mechanism. J. Mol. Biol. 376:1272-81.
Fokine, A., Bowman, V. D., Battisti, A. J., Li, Q., Chipman, P. R., Rao, V. B. and Rossmann, M. G. (2007) Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins. Virology 367:422-427.
Fuller, D. N., Raymer, D. M., Kottadiel, V. I., Rao, V. B. and Smith, D. E. (2007) Single Phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability. Proc. Natl. Acad. Sci. U.S.A. 104:16868-16873.
Sun S, Kondabagil K, Gentz PM, Rossmann MG, and Rao V. B. (2007) The Structure of the ATPase that Powers DNA Packaging into Bacteriophage T4 Procapsids. Mol Cell. 25:943-949.
Li, Q., Shivachandra, S.B., Zhang, Z. and Rao, V. B. (2007) Assembly of the Small Outer Capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid. J. Mol. Biol 370:1006-1019.
Draper, B and Rao, V. B. (2007) An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests inchworm type translocation mechanism. J. Mol. Biol. 367:79-94.
Shivachandra, S. B., Li, Q., Peachman, K. K., Matyas G. R., Leppla S. H., Alving C. R.,Rao, M., Rao V. B. (2007) Multicomponent anthrax toxin display and delivery using bacteriophage T4. Vaccine 25:1225-35.
Kondabagil,K. R., Zhang, Z. B. and Rao, V. B. (2006) The DNA translocating ATPase Of bacteriophage T4 packaging motor. J. Mol. Biol. 363:786-799.
Li, Q., Shivachandra, S., Leppla, S. H. and Rao, V. B. (2006) Bacteriophage T4 Capsid: Unique Platform for Efficient Surface Assembly of Macromolecular Complexes. J. Mol. Biol. 363: 577-578.
Sathaliyawala, T., Rao, M., Maclean, D. M., Birx, D. L., Alving, C. R. and Rao, V. B. (2006) Assembly of Human Immunodeficiency Virus (HIV) antigens on Bacteriophage T4: a Novel In Vitro Approach To construct Multicomponent HIV Vaccines. J. Virol. 80:7688-7698.
Kondabagil, K.R. and Rao, V.B. (2006) A Critical Coiled Coil Motif in the Small Terminase, gp16, from Bacteriophage T4: Insights into DNA Packaging Initiation and Assembly of Packaging Motor. J. Mol. Biol. 358:67-72.
Mitchell, M. and Rao, V.B. (2006) Functional Analysis of the Bacteriophage T4 DNA Packaging ATPase Motor. J. Biol. Chem. 281:518-527.
Rao, V.B. and Black L.W. (2005) Bacteriophage T4 DNA Packaging, in Viral Genome Packaging, Ed.C. Catalano, Landes Biosciences pp40-58.
Kanamaru S, Kondabagil K, Rossmann MG, Rao V.B. (2004) The functional domains of bacteriophage T4 terminase. J Biol Chem. 279:40795-801.
Mitchell, M. and Rao, V. B. (2004) Novel and Deviant Walker A ATP-binding motifs in Bacteriophage Large Terminase/DNA Packaging Proteins. Virology 321:217-221.
Fokine, A., Chipman, P., Leiman, P., Mesyanzhimov, V., Rao, V. B. and Rossmann, M., (2004) Molecular architecture of the prolate head of bacteriophage T4. Proc. Natl. Acad. Sci. USA 101:6003-6008.
Mitchell, M., Matsuzaki, S., Imai, S. and Rao, V. B. (2002) Sequence analysis of bacteriophage T4 DNA-packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases. Nucleic Acids Res. 30: 4009-4021.