Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly

Abstract

Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of E. coli bacteriophage HK97 and other phages, sixty capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the “E-loop,” that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit’s backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal size capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly.

Of capsid structure and stability: The partnership between charged residues of E-loop and P-domain of the bacteriophage P22 coat protein

“…Residue W61 at the tip of the E-loop was recently identified as important for both intra-and inter-capsomer hydrophobic interactions (Asija and Teschke, 2019). In addition, a recent 3.3 Å structure of P22 virions showed several salt bridges between residues of the E-loop of one subunit and the P-domain of an adjacent subunit in a capsomer that were suggested to play an important stabilizing role for P22 capsids, in a fashion similar to those seen with phage HK97 (Hasek et al, 2017;Hryc et al, 2017). Here we have mutagenized these residues and analyzed the effects of the substitutions on procapsid and capsid stability, and assembly, both in vivo and in vitro.…”

“…Beyond these crosslinks, ionic interactions between other amino acyl residues of HK97 coat proteins are important for assembly. For instance, reside E153 of the E-loop makes a stabilizing salt bridge with R210 of an adjacent subunit within a capsomer (Hasek et al, 2017). When either residue is mutated, aberrant assemblies form because the capsomers are unable to maintain the correct dome-shape required for assembly into proper procapsids and instead form sheets or tubes (Hasek et al, 2017).…”

“…For instance, reside E153 of the E-loop makes a stabilizing salt bridge with R210 of an adjacent subunit within a capsomer (Hasek et al, 2017). When either residue is mutated, aberrant assemblies form because the capsomers are unable to maintain the correct dome-shape required for assembly into proper procapsids and instead form sheets or tubes (Hasek et al, 2017). There are also electrostatic interactions that staple together subunits between and within capsomers (Tso et al, 2014).…”

A hydrophobic network: Intersubunit and intercapsomer interactions stabilizing the bacteriophage P22 capsid

“…During maturation, the hexons rearrange so they become symmetric and flattened, leading to thinner walls in the virion. In addition, many inter-subunit contacts along the periphery of the A-domain must be broken and new ones formed during maturation (28, 29), so other capsomer contacts must remain intact for the particles to survive maturation.…”

A Hydrophobic Network: Intersubunit and Intercapsomer Interactions Stabilizing the Bacteriophage P22 Capsid

“…During maturation, the hexons rearrange so they become symmetric and flattened, leading to thinner walls in the virion. In addition, many intersubunit contacts along the periphery of the A domain must be broken and new ones formed during maturation (28,29), so other capsomer contacts must remain intact for the particles to survive maturation.…”