Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly

Figures

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  FIG 1. HSV-1 capsid assembly starts with the cleavage of the scaffold and angularization of the spherical, instable procapsids into three stable capsid types resulting from a mismatch in timing between scaffold digestion and DNA packaging: scaffold-containing B-capsids; empty A-capsids, which initiated the DNA packaging process but failed to retain the genome; and DNA-filled C-capsids. Only C-capsids turn into infectious virions after recruitment of tegument proteins and lipid envelopment.

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  FIG 2. (A) 3D cryo-electron microscopy reconstruction of wild-type C-capsids imaged within intact HSV-1 virions (EMD-6386 (52)) with five copies of pUL25 (PDB 2F5U (26)) fit around each capsid penton. (B) The binding locations of pUL25 (N and C termini and the approximate amino acid positions are indicated) and other CVSC proteins are shown in the top and side view cartoons.

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  FIG 3. Identification of the CVSC subunits. (A) Coomassie-stained gels of WT (KOS HSV-1) and UL25-null (Δ25) capsids demonstrate approximately equal amounts of the sample according to capsid protein bands. (B) Western blots showing UL25 and UL17 on WT and UL25-null capsids. Data were adopted from our previous publication (3).

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  FIG 4. (A) Cryo-EM reconstruction (EMD-1354 (54)) is colored to indicate the positions of UL25 (orange) in relation to the penton (black pentagon outline). UL25 connects capsomers by binding with the penton and the underlying triplexes (blue on reconstruction). Neighboring hexons are outlined in green. (B) A high-resolution AFM image of HSV-1 A-capsid with a penton (black) and its five neighboring hexons (green) are indicated. (C) AFM image of intact HSV-1 A-capsid in liquid. The inset shows a topographical profile. (D) AFM image and topographical profile of the same HSV-1 A-capsid after breaking with nano-indentation. (E) Force-distance (FZ) curves for glass substrate and HSV-1 A-capsid. Capsid breaking is observed as a drop in the force curve. FZ curve allows direct measurement of Fbreak and k values. The k values are quantified by fitting Gaussian functions to histograms of multiple measurements of unique capsids. (F) Representative histograms of spring constants, k, for HSV-1 WT A-capsid versus UL25-null A-capsid showing a weakening of the A-capsid with deleted UL25.

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  FIG 5. Spring constant, k, (Fig. 4A) and breaking force, Fbreak, (Fig. 4B) reflect the mechanical stability of HSV-1 UL25 mutant capsids as a function of the number of amino acids (aa) in UL25 before the stop codon (s) (WT UL25 has 580 aa). For both B-capsids and A-capsids, spring constant (top) and breaking force (bottom) showed a significant increase when UL25 aa length is increased from aa 0 (UL25-null) to aa 104. For B-capsids, both breaking force and stiffness remain constant when aa length of UL25 is further increased from aa 104 to aa 580. For A-capsids, while the spring constant is unchanged for UL25 mutants with aa 104 to 580, the breaking force continued to rise with increasing aa of UL25 up to aa 577. UL25 aa 1 to 577 mutant had the same capsid stability as WT UL25 capsid with 580 aa UL25.

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  FIG 6. Many of the characterized UL25 mutations can be divided into three types. The first represents mutations that inhibit UL25 binding to capsids and result in a loss of infectivity. These mutations typically map to the N-terminal domain of UL25, which is essential for capsid interaction. Because these mutants do not bind capsids, their stability would be identical to UL25-null capsids. The second type of mutants binds capsids but does not retain packaged genomes. The third type consisted of a mutant that was missing the last three amino acids of UL25. This UL25 mutant Δ578-580 (UL25 aa 1 to 577) binds to capsids, and the capsids retained packaged genomes forming C-capsids but failed to produce infectious viruses. All three types of mutants were used to investigate the role of UL25 in capsid reinforcement. Coils in the C-capsids represent packaged DNA while A- and B-capsids are empty. Table 1 shows the list of HSV-1 UL25 mutants analyzed with AFM nano-indentation assay.