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Dec. 19, 2001 Journal Club Nozomi Ando

“The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure” Gaspar, L.P., Terezan, A.F., Pinheiro, A.S., Foguel, D., Rebello, M.A., and Silva, J. L., J. Bio. Chem. 276 , 10 (2001) 7415-7421. Dec. 19, 2001 Journal Club Nozomi Ando. Introduction: Viruses.

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Dec. 19, 2001 Journal Club Nozomi Ando

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  1. “The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure”Gaspar, L.P., Terezan, A.F., Pinheiro, A.S., Foguel, D., Rebello, M.A., and Silva, J. L., J. Bio. Chem.276, 10 (2001) 7415-7421. Dec. 19, 2001 Journal Club Nozomi Ando

  2. Introduction: Viruses Barley Yellow Mosaic Virus Bacteriophage T4 Ebola Zaire Influenza

  3. Basic Structure of Viruses VIRION: virus particle NUCLEIC ACID CORE: single- or double-stranded DNA or RNA CAPSID: protein shell that encloses the nucleic acid core; NUCLEOCAPSID: capsid and nucleic acid core ENVELOPE: lipid/protein membrane covering nucleocapsids of some kinds of viruses HIV virion

  4. Enveloped Viruses Envelope: common in many animal viruses; viral membranes are very similar to host cell membrane. Infection: viral membrane fuses to the host cell membrane, releases genome into cell. Release of new virions from host: budding through host membrane. HSV envelope breaking, revealing nucleocapsid HIV virions budding from host cell

  5. Stability of Nucleocapsids of Enveloped Viruses Outside host cell, virions must be very stable. BUT... Inside host cell, capsids must easily break down to release nucleic acid. Q: How does the nucleocapsid of enveloped viruses work? A: Many proteins are known to denature under pressure. Let us probe the effect of pressure on capsid proteins with and without the envelope.

  6. Experimental Procedure MATERIALS: enveloped Mayaro (MV) and non-enveloped Mayaro (MV-NC) PARAMETERS: hydrostatic pressure, [urea] Gel electrophoresis: MV-NC lacks envelope glycoproteins (P1 and P2) Lane 1: MV Lane 2: MV-NC

  7. Pressure Effects A: Tryptophan fluorescence measures local changes in environment of Trp residues in proteins. Change from initial state is less and mostly reversible for MV, while greater and mostly irreversible for MV-NC. B: Light scattering measures dissociation of virions. MV does not dissociate significantly. MV-NC dissociates irreversibly. MV MV decompressed MV-NC MV-NC decompressed

  8. [Urea] Effects Trp fluorescence and light scattering show that while MV seems to be slightly more resistant, both MV and MV-NC undergo structural changes as [urea] increases. At 8.0 M urea, both MV and MV-NC are far from their initial state. MV MV-NC

  9. Pressure- and Urea-Induced Disassembly A: Fluorescence emission of MV-NC at 2.5 kbar is very close to that of MV-NC in 8.0 M urea. MV-NC is disassembled by pressure. B: Fluorescence emission of MV at 2.5 kbar is closer to that of MV at atmospheric pressure than that of MV in 8.0 M urea. MV is not as easily disassembled by pressure. Atmospheric pressure 2.5 kbar 8.0 M urea

  10. Pressure Effects: Exposure of Nucleic Acid Ethidium bromide binds to RNA. EB Fluorescence determines amount of nucleic acid exposed. As pressure is increased, more RNA is exposed from MV-NC, while there is little change in the amount of RNA exposed from MV. Pressure not only deforms non-enveloped capsids, but also releases nucleic acid. MV MV decompressed MV-NC MV-NC decompressed

  11. Proteins Affected by Pressure Bis(8-anilinonapthalene-1-sulfonate) (bis-ANS) binds to partially folded proteins and nucleic acid binding sites in capsid proteins. A: High bis-ANS binding in MV-NC after decompression implies that capsid protein-RNA interactions are broken. B: High bis-ANS binding in MV at 2.5 kbar implies that glycoproteins in membrane suffer structural changes with pressure. MV loses infectivity after pressurization. (a) 1 bar, (b) 2.5 kbar, (c) returning to 1 bar, (d) 8.0 M urea

  12. Conclusion • MV-NC readily dissociates irreversibly and releases RNA at high pressures. • MV loses infectivity at high pressure but has been shown to suffer less structural changes, which are mostly reversible. • Capsid proteins, which alone disassemble under high pressure, are made stable by the existence of the envelope. The removal of the envelope puts the nucleocapsid in a metastable state. • The envelope acts as a “Trojan horse”, which allows fusion to the host cell and release of the nucleocapsid into the cell. The released nucleocapsid is pushed into a metastable state, which allows for the release of the nucleic acid.

  13. References 1. Gaspar, L.P., Terezan, A.F., Pinheiro, A.S., Foguel, D., Rebello, M.A., and Silva, J. L., J. Bio. Chem.276, 10 (2001) 7415-7421. 2. The Big Picture Book of Viruses http://www.tulane.edu/~dmsander/Big_Virology/BVHomePage.html 3. MIT Biology Hypertextbook http://esg-www.mit.edu:8001/esgbio/7001main.html

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