Análisis estructura-función de interfases proteína-proteína en un virus esférico, el virus diminuto del ratón

Abstract

The assembly and stability of a nonenveloped spherical viflon must depend on the establishment of a number of noncovalent interedions between the pmtein subunits that fonn its capsid. lnteractions between the capsid and the enclosed viral nucleic acid could also contribute to the stabili of the virion. This doctoral thesis presents the results of a thomugh mutational analysis of the pmtein-pmtein interfaces between the subuna of a viral capsid, and of the visible protein-nucleic acid interfaces in the virion. As a model we have used the pawovirus minute virus of mice, one of the structurally simplest spherical virions known. The analysis has allowed to identify amino acid residues at the pmtein subunit interfaces that are involved in capsid assembly. stability andlor wnfomaüon, and those at the capsid-nucleic acid interfaces that are involved in nucleic acid packaging, virion stabili andlor infectivity. The qm%c resub obtained can be sumnwked as fdlows. 1) Removal of some interactions prediied to be important for holding trimers of the capsid protein together led to the isolaüon of stable trimaric structures, and wnfinned that the MVM capsid assembles through trimeric intermediates. 2) Systematic mutation to alanine of 28 selected residues at the intertrimer interfaces showed that only a few residues per subunit (W203, F247, W543, Y522, T249. Ql29, K153) are individually critica1 for MVM capsid assembly. All but one of these residues wrrespond to those involved in the highest number of hydrophobic contacts or in hydmgen bonds or salt bridges between trimers. The vast majorily of the interfacial residues are not individually needed for capsid assembly or contnbute substantially to capsid stabili against dissociation. 3) The interfacial residues located around the base of the pores at the 5-fold symmetry axes of the capsid 0140. S43. N149. N170. D263, F526) are also needed for viml infectivity. These residues are invoived in a wnfonnaüonal change of the capsid that is associated with the extemaliuation of the N-tenninal segment of capsid pmtein VP2. 4) Other residues at the intertrimeric interfaces that are not critica1 for capsid assembly or stability are, however. involved in virus viability. Together. the residues that delimit the intersubunit interfaces in a spherical virus capsid are involved in several functions naeded for the completim of the viral lii cyde. 5) The involvement of capsid residues that interact with the viral nucleic acid on the stabili and function of a spherbl virus has been establihed. Several amino acid residues at the capid-DNA interfaces are invdved in the wnfonnatimal stabilii of the MVM virion. 6) One residue (D273) in each capsid subunit, negativdy chaged and located at Me interna1 suríace of the capsid, 1s involved in the packaging of the viral DNA inside the MVM virion.