Quantification of the DNA cleavage and packaging proteins U(L)15 and U(L)28 in A and B capsids of herpes simplex virus type 1

Abstract

The proteins produced by the herpes simplex virus type 1 (HSV-1) genes U(L)15 and U(L)28 are believed to form part of the terminase enzyme, a protein complex essential for the cleavage of newly synthesized, concatameric herpesvirus DNA and the packaging of the resultant genome lengths into preformed capsids. This work describes the purification of recombinant forms of pU(L)15 and pU(L)28, which allowed the calculation of the average number of copies of each protein in A and B capsids and in capsids lacking the putative portal encoded by U(L)6. On average, 1.0 (+/-0.29 [standard deviation]) copies of pU(L)15 and 2.4 (+/-0.97) copies of pU(L)28 were present in B capsids, 1.2 (+/-0.72) copies of pU(L)15 and 1.5 (+/-0.86) copies of pU(L)28 were found in mutant capsids lacking the putative portal protein pU(L)6, and approximately 12.0 (+/-5.63) copies of pU(L)15 and 0.6 (+/-0.32) copies of pU(L)28 were present in each A capsid. These results suggest that the packaging machine is partly comprised of approximately 12 copies of pU(L)15, as found in A capsids, with wild-type B and mutant U(L)6(-) capsids containing an incomplete complement of cleavage and packaging proteins. These results are consistent with observations that B capsids form by default in the absence of packaging machinery in vitro and in vivo. In contrast, A capsids may be the result of initiated but aborted attempts at DNA packaging, resulting in the retention of at least part of the DNA packaging machinery.

FIG. 1.

Scanned digital image of Coomassie-stained gels showing protein profiles from relevant steps of the purification of U_L15 (A) and U_L28 (B) proteins. (A) Lane 1, initial cell lysate; lane 2, supernatant after lysis; lane 3, supernatant from wash of insoluble material; lane 4, eluted protein after dialysis. (B) Lane 1, initial cell lysate; lane 2, supernatant after lysis; lane 3, Ni-nitrilotriacetic acid agarose beads after incubation with the insoluble, denatured protein fraction and extensive washing; lane 4, supernatant from wash of agarose beads; lane 5, eluted protein after dialysis. Molecular mass standards are indicated on the left in kilodaltons.

FIG. 1.

Scanned digital image of Coomassie-stained gels showing protein profiles from relevant steps of the purification of U_L15 (A) and U_L28 (B) proteins. (A) Lane 1, initial cell lysate; lane 2, supernatant after lysis; lane 3, supernatant from wash of insoluble material; lane 4, eluted protein after dialysis. (B) Lane 1, initial cell lysate; lane 2, supernatant after lysis; lane 3, Ni-nitrilotriacetic acid agarose beads after incubation with the insoluble, denatured protein fraction and extensive washing; lane 4, supernatant from wash of agarose beads; lane 5, eluted protein after dialysis. Molecular mass standards are indicated on the left in kilodaltons.

FIG. 2.

Data used for calculating the average number of copies of pU_L15 or pU_L28 in capsids. (A) Coomassie-stained gel with B capsids (lanes 1 and 2) and BSA standards (lanes 3 to 8). The amount of BSA (in nanograms) loaded is indicated at the bottom of lanes 3 to 8. The R² value of the graph produced by analysis of the data was 0.98, and the estimated amount of VP5 (arrow) is indicated in italics below lanes 1 and 2. The positions of molecular weight standards are indicated on the left. (B) Immunoblot of B capsids (lanes 1 and 2) and purified U_L15 protein standards (lanes 3 to 8) probed with antisera directed against pU_L15. The amount of pU_L15 (in nanograms) is indicated below each lane. (C) Immunoblot probed with antisera directed against pU_L28. B capsids are in lanes 1 and 2, and purified U_L28 protein standards are in lanes 3 to 9. The amount of pU_L28 (in nanograms) is indicated below each lane.

FIG. 2.

Data used for calculating the average number of copies of pU_L15 or pU_L28 in capsids. (A) Coomassie-stained gel with B capsids (lanes 1 and 2) and BSA standards (lanes 3 to 8). The amount of BSA (in nanograms) loaded is indicated at the bottom of lanes 3 to 8. The R² value of the graph produced by analysis of the data was 0.98, and the estimated amount of VP5 (arrow) is indicated in italics below lanes 1 and 2. The positions of molecular weight standards are indicated on the left. (B) Immunoblot of B capsids (lanes 1 and 2) and purified U_L15 protein standards (lanes 3 to 8) probed with antisera directed against pU_L15. The amount of pU_L15 (in nanograms) is indicated below each lane. (C) Immunoblot probed with antisera directed against pU_L28. B capsids are in lanes 1 and 2, and purified U_L28 protein standards are in lanes 3 to 9. The amount of pU_L28 (in nanograms) is indicated below each lane.

FIG. 3.

Immunoblots of B capsids purified through two sequential 20- to-50% sucrose gradients. The 14-ml gradient was fractionated from the top (fraction one), and the proteins present in each fraction were separated on an 8% denaturing polyacrylamide gel before being transferred to a PVDF membrane. The membrane was then probed with antisera against U_L28, U_L15, or VP5 and developed using the ECL+ method (see Materials and Methods). The image was generated using a Molecular Dynamics Storm PhosphorImager with chemiluminescence detection capability.

FIG. 4.

Nuclear lysate of Vero cells infected with the U_L18 deletion virus K23Z, purified on two sequential 20- to-50% sucrose gradients. The 14-ml gradient was fractionated from top (lane 1) to bottom (lane 11), and the proteins present in each fraction were separated on an 8% denaturing polyacrylamide gel before being transferred to nitrocellulose membrane. The membrane was then probed with antisera against U_L15 (A) or U_L28 (C) and developed using the ECL+ method (see Materials and Methods). Lane 12 contains purified protein as a positive control (arrow). As a further control, lysates of HSV-1(F)-infected, K23Z-infected, or mock-infected cells were electrophoretically separated and reacted with antisera against U_L15 (B) or U_L28 (D) to confirm expression of the proteins in the cells infected with mutant virus.

FIG. 5.

Coomassie-stained gel of A and B capsids purified as described in Materials and Methods. The positions of the capsid shell proteins are indicated on the right. Note the very small amount of VP22a present in the A capsid sample compared to that in B capsids.

FIG. 6.

Digital image of immunoblot probed with antisera directed against the portion of the U_L15 protein encoded by the entire exon II of the gene (A) or the N-terminal 35 amino acids (B). Lanes 1 and 2, A capsids; lanes 3, 4, and 5, B capsids; lanes 6 and 7, U_L6(−) capsids. All capsids were purified through two successive sucrose gradients (see Materials and Methods). The 83,000 and 80,000 apparent M_r forms of pU_L15 are indicated with arrows.