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J. Biol. Chem., Vol. 280, Issue 18, 17969-17977, May 6, 2005

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Functional Relevance of Amino Acid Residues Involved in Interactions with Ordered Nucleic Acid in a Spherical Virus^*

Juan Reguera, Esther Grueso, Aura Carreira, Cristina Sánchez-Martínez, José M. Almendral, and Mauricio G. Mateu

From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid, Spain

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.

Received for publication, January 24, 2005 , and in revised form, February 22, 2005.

^* This work was supported by independent grants from Ministerio de Ciencia y Tecnología and Comunidad Autónoma de Madrid (to the laboratories of M. G. M. (Grants BIO2003-04445 and 07B/0012/2002) and J. M. A. (Grants SAF2001-1325 and 07B/0020/2002)), by EU contract QLK3-CT-2001-01010 (to J. M. A.), and by an institutional grant from Fundación Ramón Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 34-91-4978462; Fax: 34-91-4974799; E-mail: mgarcia{at}cbm.uam.es.

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