M.M. Hernandez

Tobacco mosaic virus

Tobacco mosaic virusTMV

• Genus Tobamovirus

15 members

naked, rigid rod,

+ unsegmented ss RNA

• No family assignation by ICTV

30 nm Helical symmetry

RNA genome

18 nm 2 nm

NC protein

STRUCTURAL FEATURES OF TMV

CAPSID

Images:P. Sforza, 2001

www.ppws.vt.edu/~sforza/tmv/tmv.html

TMV genome organization

5’ cap

6,395 nt

30 K

MT-Hel

UAG (leaky)

183K

Replicase RdRp

tRNAhis17.6 K

Movement MP

Capsid CP

126 K

Host: Nicotiana tabaccum (Solanaceous ) & other 9 plant families

susceptible too

Disease:Mosaic (calico)

Mosaic disease on Tobacco leaf

Necrosis Necrosis

Chlorosis StuntingStunting

Mosaic disease: significant losses in yield & $ value

TMV Life cycle a) Virus entry trough abrasions on plant tissue. Inside cell associates with ER

b) spontaneous release of few capsid (CP) subunits 5' end of genome is uncovered

c) Host ribosome attaches to viral RNA, moves down displacing more CP units

5’ cap Host Rb

…/…

Modification of a diagram from: L. Stannard, Department of Medical Microbiology, U. of Cape Town

d) Ribosome meets start codon, translates first two proteins (126K ,183 K) while uncoating continues “co-traslational disassembly” e) 126 K ( MET-Hel) & 183 K ( RdRp) use viral RNA as template to make full length complementary neg. strand RNA

TMV Life cycle

(cont.) 5’

…/…

Modification of a diagram from: L. Stannard, Department of Medical Microbiology, U. of Cape Town

TMV Life cycle

(cont.) f) Neg. RNA strand used by viral replicase (RdPp/MET-Hel ) as template for +RNA

g) Also, neg. RNA strand has internal promoters used by replicase to make mRNA for 30K protein (MP) and 17.5 K (CP)

RdRp + Strand (genome)

Neg. strand

promoters

Transcription by RdRp

MP mRNACP mRNA

TMV Life cycle

(cont.)

h) MP combines with viral +RNA to move it into new plant cells through plasmodesmata

http:www.sunysb.edu/biochem/BIOCHEM/facultypages/citovsky/TMV-TransportModel-III2.jpg

TMV Life cycle

(cont.) i) Accumulation of +RNA & CP proteins stimulates assembly of progeny virions

CP units self assemble into discs

Viral RNA interacts with discs

Electrostatic interaction stabilizes structure into helix

M. Olvera de la Cruz

TMV Life cycle

(cont.) j) massive TMR replication occur in the X-bodies (viroplasmas)

Hamacher et al. 2003

Szecsi et al. 1999

TMV Life cycle

(cont.) k) Virus will remain in plant tissue and sap until delivered to a new plant host usually by mechanical means

TMV diagnosis

• Symptoms

• E.M. & Serology

• RT-PCR & RFLP

HTTP;//BAKERLAB.BERKELEY.EDU/?IMG=1Letschert et al. 2002

• Improving diagnostic techniques

• Characterization of MP and viral movement through plant

• Characterization of the Helicase & RdRp

Current Research on TMV

Questions?

Agrios G. N. 1988. Plant Pathology Third Edition. Academic Press, Inc. San Diegopp: 658663Ashby J., E. Boutant, M. Seemanpillai, A. Sambade, C. Ritzenthaler, M. Heinlein . 2006. Tobacco Mosaic Virus Movement Protein Functions as a structural Microtubule-Associated Protein. Journal of Virology. 80(17):8329-8344Bos L. 1999. Plant Viruses Unique and intriguing pathogens. Backhuys Publishers LeidenDovas C., K. Efthimious, N. I. Katis. 2004. Generic detection and differentiation of tobamoviruses by a spot nested RT-PCR-RFLP using dI-contining primers along with homologous dG-containing primers. Journal of Virological Methods. 117:137-144 Goelet P., Lomonossoff G. P., Blutler P. JG, Akam M. E., Gait M. J., and Karn J ., 1982. Nucleotide sequence of the tobacco mosaic virus RNA. Proc. Natl. Acad. Sci. U.S.A. 79: 5818-5822Goregaoker S. P. and J. N. Culver. 2003. Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins. Journal of Virology. 77 (6): 3549- 3556Hirashima K. and Y. Watanabe. 2001. Tobamovirus Replicase Coding Region is Involved in Cell-to-Cell Movement. Journal of Virology. 75 (18) 8831-8836Hirashima K. and Y. Watanabe. 2003. RNA Helicase Doamin of Tobamovirus Replicase Executes Cell-to-Cell Movement Possibly through Collaboration with its Nonconserved Region. Journal of Virology. 77 (22) 12357- 12362Horst K.R.(reviser), 2001 Westcott=s Plant Disease Handbook Six Edition. Kluwer Academic Plublishers.Boston pp:511-513Jacobi V., G.D. Bachand, R.C. Hamelin and J.D. Castello. 1998. Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses. Journal of Virological Methods 74 (2):167-178 International Committee on Taxonomy of Viruses (ICTV) 2002. ICTVdB Virus Descriptions. 71.0.1.0.001 Tobacco mosaic virus (vulgare strain; ssp. NC82 strain). Retrieved November 22, 2002, from: http://www.ncbi.nih.gov/ICTVdb/ICTVdB/71010001.htm Kawakami F. M., R.W. Kim, R.N. Beachy. 2006 Domains of tobacco mosaic virus movement protein essential for its membrane association. Journal of General Virology 87(9):2699-7707

REFERENCES

Khan J. A. and J. Dijkstra editors, 2002. Plant Viruses as Molecular Pathogens Food Product Press 7 An Imprint of the Haworth Press Inc. New YorkLazarowitz S. G., 2001. Plant Viruses In: Fundamental Virology Fourth Edition. Knipe D. and P.M. Howley Editors. Lippincott Williams & Wilkins, Philadelphia. pp:377-429 Lesemann D.-E., S. Winters. 1998. Detection and specific identification of tobamoviruses in horticultural crops by immunoelectron microscopical methods (IEM) and ELISA. Mitteilungender Biologischen Bundesanstalt fur Land-und Forstwirtschaft 357Letschert B., A Gunter, L. Dietrich-Eckhardt, P. Willingmann, C. Heinze., 2002. Detection and differentiation of serologically cross-reacting tobamoviruses of economical importance by RT-PCR and RT-PCR-RFLP. Journal of Virological Methods 106:1-10 Lucas G. B. , C.L. Campbell, and L.T. Lucas, 1985. Introduction to Plant Diseases Identification and Management. The AVI Publishing Company, Inc. Westport Connecticut. pp 254-261Martin J.H. , W.H. Leonard and D. L. Stamp 1976 Principles of Field Crop Production Third Edition MacMills Publishing Co., INC. LondonMatthews R.E.F., 1992. Fundamentals of Plant Virology Academic Press Inc San DiegoMatthews R.E.F., 1999 Plant Virology Third Edition. Academic Press Inc San Diego. pp:207-225Osman T. A. M., and K.W. Buck. 2003 Identification of a Region of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replication Proteins Which bind Specifically to the Viral 3'-Terminal tRNA-like Structure. Journal of Virology 77(16 ) 8669-8675Padmanadban M.S., Goregaoker S.P., S. Golem, H. Shiferaw, and J. N. Culver . 2005. Interaction of the Tobacco Mosaic Virus Replicase Protein with the Aux/IAA Protein PAP1/IAA26 Is associated with Disease Development. Journal of Virology. 79 (4) 2549 - 2558Sacristan S., J. M. Malpica, A. Fraile, F. Garcia-Arenal. 2003. Estimation of Population Bottlenecks during Systemic Movement of Tobacco Mosaic Virus in Tobacco Plants. Journal of Virology 77(18) 9906-9911Seemanpillai M., R. Elamawi, C. Ritzenthhaler, M. Heinlein. 2006. Challenging the Role of Microtubules in Tobacco Mosaic Virus Movement by Drug Treatment is Disputable. Journal of Virology 80(13): 6712-6715 Torok V.A. and J.W. Randles. 2001. Tobacco mosaic virus RNA as an internal control for duplex RT-PCR assay of pea germaplsm. Australian Plant Pathology 30: 227-230Zaitlin M., 2001. Tobamovirus in: The Spriger Index of Viruses. Tidona C. A. and G. Darai Editors. Springer-Verlag, Berlin

1. Solution of TMV Capsid Proteins

Salt (M)

Acidity

Electrostatic repulsion inhibits + protein +protein aggregation.

2. Add ssTMV+ RNA (assembly infective virus): Electrostatic attraction stabilizes viral assembly.

ZRNA= -1/mer

4. Capsid + Hole (density ≈ RNA crystal) self-assembles from capsid protein + viral RNA solution

3. Sphere-like (icosahedral) RNA virus(Tsuruta et al)