RESEARCH INTERESTS:

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.

RESEARCH FUNDING:

 Our research is funded by the National Science Foundation and the National Institutes of Health.

RECENT PUBLICATIONS:

1.  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.

2.   Rao, V.B. and Feiss, M. The bacteriophage DNA packaging motor. (2008) Ann. Rev.   Genetics  42:642-681.

3.  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.

4.   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.

5.  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.

6.  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.

7.  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.

8.  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.

9.  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 bacteiophage T4. Vaccine 25:1225-35.

10. 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.

11. 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.

12. Sathaliyawala, T., Rao, M., Maclean, D. M., Birx, D. L., Alving, C. R. and Rao, V. B. (2006)  Assembly og Human Immunodeficiency Virus (HIV) antigens on Bacteriophage T4: a Novel In Vitro Approach To Construct Multicomponent HIV Vaccines. J. Virol. 80:7688-7698.

13. 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.

14. Mitchell, M. and Rao, V.B. (2006) Functional Analysis of the Bacteriophage T4 DNA Packaging ATPase Motor.  J. Biol. Chem. 281:518-527.

15. Rao, V.B. and Black L.W. (2005) Bacteriophage T4 DNA Packaging, in Viral Genome Packaging, Ed. C. Catalano, Landes Biosciences pp40-58.

16. Kanamaru S, Kondabagil K, Rossmann MG, Rao V.B. (2004) The functional domains of bacteriophage T4 terminase. J Biol Chem. 279:40795-801.

17. 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.

18. 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.

19. 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.