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Journal of Virology, June 2007, p. 6231-6240, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02859-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Location and Role of Free Cysteinyl Residues in the Sindbis Virus E1 and E2 Glycoproteins{triangledown}

Christopher B. Whitehurst, Erik J. Soderblom, Michelle L. West, Raquel Hernandez, Michael B. Goshe, and Dennis T. Brown*

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695

Received 26 December 2006/ Accepted 21 March 2007

Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.

* Corresponding author. Mailing address: North Carolina State University, Department of Molecular and Structural Biochemistry, 128 Polk Hall, Raleigh, NC 27695. Phone: (919) 515-5765. Fax: (919) 515-2400. E-mail: dennis_brown{at}ncsu.edu

{triangledown} Published ahead of print on 4 April 2007.

Journal of Virology, June 2007, p. 6231-6240, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02859-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hafer, A., Whittlesey, R., Brown, D. T., Hernandez, R. (2009). Differential Incorporation of Cholesterol by Sindbis Virus Grown in Mammalian or Insect Cells. J. Virol. 83: 9113-9121 [Abstract] [Full Text]  


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