Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

Abstract

Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles. P rotein-protein recognition mediates many fundamental biological processes. A detailed knowledge of these processes requires the determination of the structural, energetic, and functional roles of individual amino acid residues and interactions in protein-protein interfaces. These studies have been generally undertaken by using small protein-ligand complexes or oligomeric proteins of moderate size (reviewed in ref. 1; see also refs. 2-4). In contrast, for multimeric protein complexes, such as viral capsids (5, 6) or large cellular assemblies, little is known about the specific molecular determinants of protein association and stability. Mutational studies of virus capsids, generally focused on a few specific amino acid residues, have provided important insights (7-22). However, exhaustive experimental studies on the relative importance of residues and molecular interactions in viral capsid assembly, disassembly, and͞or stability are still very limited. These studies contribute also to the understanding of protein structure-function relationships and evolution under conflictive selective constraints (22-27), and they could be exploited possibly in the design of thermostable vaccines and antiviral agents promoting capsid disassembly or interfering with assembly (23, 28-31).Many viruses, including viruses of medical or veterinary significance, have capsids of icosahedral symmetry. The icosahedral T ϭ 1 capsids of parvoviruses (32-37) are formed by 60 protein subunits that are contributed by three nonidentical polypeptide chains (VP1, VP2, and VP3). These polypeptides derive, however, from a single gene and show identical fold and core sequence (Fig. 1). In the minut...

Nuclear Export of the Nonenveloped Parvovirus Virion Is Directed by an Unordered Protein Signal Exposed on the Capsid Surface

“…Thus, intracellular structural transitions of the MVM capsid configuration may trigger an alternate exposed outside of the shell across the channel at the fivefold axis (1, 68) of the nuclear transport signals localized at the N termini of the VP protein subunits, driving the traffic of the virus through the NPC. In support of this model, mutations of the interfacial amino acid residues surrounding the base of the channel hampered MVMp infectivity (55).…”

Mutational Analysis of Narrow Pores at the Fivefold Symmetry Axes of Adeno-Associated Virus Type 2 Capsids Reveals a Dual Role in Genome Packaging and Activation of Phospholipase A2 Activity

“…Mutational analysis of AAV2 capsid proteins revealed that mutations within the beta-barrel structure and at the very N or C terminus of VP3 impaired assembly (53,72,80). It was shown for MVM that a few residues involved in intertrimer interactions have a crucial role in capsid assembly and stability (51). In cells expressing CPV proteins, small amounts of trimers could be detected (85).…”

“…Several observations with autonomous parvoviruses also support this interpretation. It has been shown for MVM that mutants with substitutions of residues close to the fivefold pores were not able to undergo the conformational transition induced by mild heating, which normally leeds to exposure of VP2 N termini on wt VP2 capsids only (51). The fivefold pores are partially filled in almost all of the resolved structures of parvoviruses (2,3,40,83).…”

Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps

“…Data based on autonomous parvoviruses suggest that, prior to escaping the endosome, the virion is thought to undergo conformational changes, leading to the exposure of the unique N terminus of Vp1, necessary for escape ( Fig. 8III) (5,33). However, exposure of Vp1 may not be the only factor involved in escape from the endosome, as was indicated in a recent study with CPV (38).…”