The herpes simplex virus 1 UL17 protein is the second constituent of the capsid vertex-specific component required for DNA packaging and retention

Abstract

The herpes simplex virus (HSV) UL17 and UL25 minor capsid proteins are essential for DNA packaging. They are thought to comprise a molecule arrayed in five copies around each of the capsid vertices. This molecule was initially termed the "C-capsid-specific component" (CCSC) (B. L. Trus et al., Mol. Cell 26:479-489, 2007), but as we have subsequently observed this feature on reconstructions of A, B, and C capsids, we now refer to it more generally as the "capsid vertex-specific component" (CVSC) (S. K. Cockrell et al., J. Virol. 85:4875-4887, 2011). We previously confirmed that UL25 occupies the vertex-distal region of the CVSC density by visualizing a large UL25-specific tag in reconstructions calculated from cryo-electron microscopy (cryo-EM) images. We have pursued the same strategy to determine the capsid location of the UL17 protein. Recombinant viruses were generated that contained either a small tandem affinity purification (TAP) tag or the green fluorescent protein (GFP) attached to the C terminus of UL17. Purification of the TAP-tagged UL17 or a similarly TAP-tagged UL25 protein clearly demonstrated that the two proteins interact. A cryo-EM reconstruction of capsids containing the UL17-GFP protein reveals that UL17 is the second component of the CVSC and suggests that UL17 interfaces with the other CVSC component, UL25, through its C terminus. The portion of UL17 nearest the vertex appears to be poorly constrained, which may provide flexibility in interacting with tegument proteins or the DNA-packaging machinery at the portal vertex. The exposed locations of the UL17 and UL25 proteins on the HSV-1 capsid exterior suggest that they may be attractive targets for highly specific antivirals.

Fig. 1.

UL17-CTAP and UL17-GFP virus constructs and single-step growth curves. (A) The HSV-1 genome is represented at the top. U_L and U_S represent the long and short unique region sequences, respectively. The region of the HSV-1 genome that contains the UL17 gene is expanded below. The UL17-GFP and UL17-CTAP fusion proteins that are expressed from the vUL17-GFP and vUL17-CTAP viruses are shown. (B) Vero or G5 cells were infected with either KOS, vUL17-GFP, vUL17-CTAP, or the UL17-null (ΔUL17) virus at an MOI of 3 at 4°C for 1 h and incubated at 37°C. The cultures were harvested at the indicated times postinfection and freeze-thawed three times, and the yield of virus at each time point was determined by plaque titer determination on G5 cells.

Fig. 2.

Analysis of capsid-bound UL17-GFP and UL17-CTAP. UL17 content in capsids isolated from cells infected with KOS, vUL17-GFP, or vUL17-CTAP. (Top) Equivalent amounts of C, B, or A capsids were separated by SDS-PAGE, and the proteins were detected by Coomassie blue staining. The positions of the capsid proteins (major capsid protein VP5, triplex proteins VP19C and VP23, small capsid protein VP26, the scaffold protein VP22a, and the portal protein UL6) and the two CVSC proteins UL25 and UL17 along with the UL17-GFP and UL17-CTAP fusion proteins are indicated. Molecular mass standards are labeled on the left in kDa. (Bottom) Immunoblot analysis of KOS, vUL17-GFP, or vUL17-CTAP C, B, or A capsids with UL17, GFP, and calmodulin antibodies.

Fig. 3.

TAP purification of UL25-NTAP and UL17-CTAP. Vero cells were infected with HSV-1 recombinant viruses expressing the indicated TAP-tagged protein or with wild-type (KOS) virus. (A) Following TAP purification, SDS-PAGE analysis (silver stain) was done on the proteins eluted from the KOS, UL25-NTAP, and UL17-CTAP calmodulin column. (B) Western blots (antibodies used listed below each blot) demonstrating the presence of the TAP-tagged proteins along with proteins that copurify with the TAP-tagged proteins. Protein standards (kDa) are shown on the left of each gel or blot.

Fig. 4.

Cryo-electron microscopy of tagged capsids. Shown are micrographs of UL17-GFP (A) and wild-type (B) capsids. DNA-filled C capsids (black arrowheads and inset close-ups) are darker than, and easily distinguishable from, subset populations of A and B capsids (white and gray arrowheads, respectively). C capsids selected from each data set were subject to the three-dimensional reconstruction procedure.

Fig. 5.

Visualizing the GFP tag on UL17-GFP capsids. Density maps calculated from C capsids with GFP-labeled UL17 (A) and wild-type C capsids with unlabeled UL17 (B). Radial sections partway between spherical and icosahedral (70%) through each map, at a radius of 565 Å at the icosahedral 3-fold axis, are shown at the top, with surface-rendered views beneath. Views are along the icosahedral 2-fold axis. In the sections, a penton and its two adjacent hexons are labeled with “P” and “H,” respectively, and a CVSC density is outlined in magenta. In the UL17-GFP map section, additional density is seen coming off the CVSC and is outlined in green. Insets show close-up views of the CVSC outlined in the sections. The densities have been shaded so that the penton is blue, hexons are purple, the CVSC is magenta, and extra density in the tagged map, which we attribute to GFP, is green. Surface views of the capsid beneath the sections have been color coded as described above, with triplexes in yellow.

Fig. 6.

Close-up views of the UL17-GFP density. A penton flanked by five CVSC densities is shown for the UL17-GFP (A) and wild-type (B) density maps. Capsid subunit boundaries have been estimated and colored as for Fig. 5. The GFP density is in green and labeled with black arrows in panel A. Into this density, the crystal structure of GFP (Protein Data Bank ID 1EMA) (22) has been docked in panel C. The capsid density is semitransparent, and the GFP coordinates are in a ribbon representation, rainbow colored, with C and N termini labeled (C and N, respectively). There is a broad contact region between GFP and the CVSC, implying interactions between the GFP moiety and UL17, in addition to the covalent link, and perhaps also with UL25. GFP also contacts an adjacent hexon (black arrow). As demonstrated schematically (D), the location of GFP places protein UL17 in the CVSC density, adjacent to UL25 and in the CVSC region closest to the penton. The UL17 C terminus, tagged by GFP, appears in close proximity to UL25.

Fig. 7.

Summary of CVSC subunit tagging. Results of successful attempts to localize and orient CVSC subunits by visualizing bulk tags on HSV-1 capsids are combined in this schematic representation. Color coding of subunits is as for Fig. 5 and 6. Previously we labeled UL25 with GFP at amino acid 50 (aa 50) (4), and the location of this residue is indicated with a green cross. We also labeled the N terminus with a TAP tag (gray oval). The locations of these two tags place UL25 in the distal-most region of the CVSC relative to the penton. The tagging data also support fitting of the crystal structure of UL25 (residues 134 to 580) into the middle part of the CVSC (dashed outline). Tagging of UL17 at the C terminus with GFP localizes UL17 to the CVSC, into the penton-proximal domain. A portion of UL17 nearest to the penton may be flexible (indicated by the motion-blurred extension of UL17). Localization of the UL17 and UL25 tags allows us to predict each protein's orientation within the CVSC (as labeled by “N” and “C,” referring to the N- and C-terminal domains, respectively) with confidence for the C terminus of UL17 and N terminus of UL25 and by inference vice versa. The UL17 and UL25 proteins appear likely to be organized in a C-terminal-to-C-terminal manner.