Association of herpes simplex virus pUL31 with capsid vertices and components of the capsid vertex-specific complex

Abstract

pU(L)34 and pU(L)31 of herpes simplex virus (HSV) comprise the nuclear egress complex (NEC) and are required for budding at the inner nuclear membrane. pU(L)31 also associates with capsids, suggesting it bridges the capsid and pU(L)34 in the nuclear membrane to initiate budding. Previous studies showed that capsid association of pU(L)31 was precluded in the absence of the C terminus of pU(L)25, which along with pU(L)17 comprises the capsid vertex-specific complex, or CVSC. The present studies show that the final 20 amino acids of pU(L)25 are required for pU(L)31 capsid association. Unexpectedly, in the complete absence of pU(L)25, or when pU(L)25 capsid binding was precluded by deletion of its first 50 amino acids, pU(L)31 still associated with capsids. Under these conditions, pU(L)31 was shown to coimmunoprecipitate weakly with pU(L)17. Based on these data, we hypothesize that the final 20 amino acids of pU(L)25 are required for pU(L)31 to associate with capsids. In the absence of pU(L)25 from the capsid, regions of capsid-associated pU(L)17 are bound by pU(L)31. Immunogold electron microscopy revealed that pU(L)31 could associate with multiple sites on a single capsid in the nucleus of infected cells. Electron tomography revealed that immunogold particles specific to pU(L)31 protein bind to densities at the vertices of the capsid, a location consistent with that of the CVSC. These data suggest that pU(L)31 loads onto CVSCs in the nucleus to eventually bind pU(L)34 located within the nuclear membrane to initiate capsid budding.

Importance: This study is important because it localizes pU(L)1, a component previously known to be required for HSV capsids to bud through the inner nuclear membrane, to the vertex-specific complex of HSV capsids, which comprises the unique long region 25 (U(L)25) and U(L)17 gene products. It also shows this interaction is dependent on the C terminus of U(L)25. This information is vital for understanding how capsids bud through the inner nuclear membrane.

FIG 1

Immunoblotting of capsids purified from cells infected with HSV-1(F). Capsids of HSV-1 were purified from infected CV1 cells as described in Materials and Methods. The capsid-containing sucrose gradient was fractionated, and the proteins in each fraction were precipitated with TCA and dissolved in SDS loading buffer. Fractions 3 to 19 from the bottom to the top of the gradient were separated on a 10% SDS-polyacrylamide gel (SDS-PAGE), followed by immunoblotting with antibodies to pU_L31 and VP5 sequentially, and the results were revealed by enhanced chemiluminescence (ECL).

FIG 2

Immunoblotting of 212 S capsids. About 4 × 10⁸ CV1 cells were infected with 212 S virus (with pU_L25 truncated at codon 212) at an MOI of 5 PFU per cell. Nuclear capsids were purified as described in Materials and Methods. The capsids were separated through a continuous sucrose gradient, 0.5-ml fractions were collected with a fractionator, and proteins were precipitated with TCA and dissolved in SDS loading buffer. The samples were separated on a 10% SDS-PAGE gel, with lanes from left to right corresponding to fractions collected from the bottom to top of the gradient. The far-right lane contains unfractionated lysates from infected cells. Immunoblotting was performed with antibodies to pU_L31, pU_L17, pU_L25, VP5, and scaffold proteins VP22a and VP21, respectively, and the results were revealed by ECL.

FIG 3

Immunoblotting of 560S capsids. CV1 cells were infected with HSV-1 recombinant virus vFH418 (with pU_L25 truncated at codon 560), and capsids were purified as described in Materials and Methods. The continuous sucrose gradient containing capsids was fractionated from bottom to top, and the protein in each fraction was precipitated with TCA and dissolved in SDS loading buffer. Denatured proteins in fractions 2 to 19 and from a sample of virus-infected lysates (far right lane) were separated on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and then probed with antibodies to pU_L31, pU_L17, pU_L25, VP5, or scaffold proteins VP22a and VP21. Bound antibodies were revealed using ECL.

FIG 4

Immunoblotting of capsids of U_L25 null mutant. Capsids of HSV-1 mutant virus vJB92 (lacking the entire U_L25 ORF) were purified from infected CV1 cells on a continuous sucrose gradient as described in Materials and Methods. Proteins in each fraction were precipitated with TCA and dissolved in SDS loading buffer. Fractions 2 to 19 were collected from the bottom to the top of the gradient. Proteins within these fractions and the unfractionated lysate (far-right lane) were separated on a 10% SDS-PAGE gel, followed by immunoblotting with antibodies to pU_L31, pU_L17, VP5, or viral scaffold proteins VP22a and VP21, respectively. Bound antibodies were revealed by ECL.

FIG 5

Immunoblotting of capsids from cells infected with a mutant lacking the first 50 amino acids of pU_L25. Capsids of HSV-1 mutant virus vFH421 (lacking the codons from aa 1 to 50 of pU_L25) were purified from infected CV1 cells by continuous sucrose gradient fractionation as described in Materials and Methods. The proteins in each fraction were precipitated with TCA and dissolved in SDS loading buffer. Fractions 3 to 20 collected from the bottom to the top of the gradient and a sample of unfractionated lysate (far right lane) were separated on a 10% SDS-PAGE gel, and the presence of proteins was determined by immunoblotting using antibodies to pU_L31, pU_L25, pU_L17, VP5, or viral scaffold proteins VP22a and VP21.

FIG 6

Immunogold analysis of pU_L31 localization in infected cells. (A) HSV-1(F). (B) HSV-1 U_L31-HA. (C) HSV-1 U_L31-HA with deletion of aa 561 to 580. (D) HSV-1 U_L31-HA with U_L25 ORF deleted. Cells were infected with the indicated viruses at 5 PFU per cell. At 18 h after infection, the cells were fixed and embedded. Thin sections were reacted with HA-specific antibody to localize pU_L31. Bound antibody was revealed by goat anti-rabbit antibody conjugated to 10-nm-diameter gold beads. Sections were examined by transmission electron microscopy. Open arrows indicate the capsids labeled with gold beads, solid arrows indicate free gold beads in nuclear (Nu) plasma, and open arrowheads indicate gold beads in nuclear membrane (NM). Cyt, cytoplasm. The size standard bar is 204 nm, and capsids are approximately 125 nm in diameter.

FIG 6

Immunogold analysis of pU_L31 localization in infected cells. (A) HSV-1(F). (B) HSV-1 U_L31-HA. (C) HSV-1 U_L31-HA with deletion of aa 561 to 580. (D) HSV-1 U_L31-HA with U_L25 ORF deleted. Cells were infected with the indicated viruses at 5 PFU per cell. At 18 h after infection, the cells were fixed and embedded. Thin sections were reacted with HA-specific antibody to localize pU_L31. Bound antibody was revealed by goat anti-rabbit antibody conjugated to 10-nm-diameter gold beads. Sections were examined by transmission electron microscopy. Open arrows indicate the capsids labeled with gold beads, solid arrows indicate free gold beads in nuclear (Nu) plasma, and open arrowheads indicate gold beads in nuclear membrane (NM). Cyt, cytoplasm. The size standard bar is 204 nm, and capsids are approximately 125 nm in diameter.

FIG 7

pU_L31 association with purified B capsids. (A) Immunoblotting of purified B capsids with anti-HA antibody and anti-VP5 antibody. (B) Examples of immunogold labeling of B capsids with 10-nm-diameter gold-conjugated anti-HA antibody. (C) More than 1,000 capsids of each type were scored as labeled or unlabeled. The histogram indicates the percentage of labeled capsids under each circumstance. GraphPad software was using for statistical analyses using Fisher's exact test. *, P < 0.01.

FIG 8

Electron tomography of pU_L31 association with purified capsids. (A and B) Two density slices from 1 of the 10 electron tomograms of immunogold-labeled B capsids. The arrows point to the capsid the gold particles are bound to. The full tomogram is shown in Movie S1 in the supplemental material. (C and D) Density slice (C) and colored surface view (D) of the capsid in the red box in panel A. Segments of the capsid are shown with the attachment of the gold particle (antibody) on the pentonic vertex of the capsid. (E and F) Density slice (E) and colored surface view (F) of the capsid in the green box in panel B. In panel C or E, only the slice revealing the attachment site along with the density in between the gold particle and the capsid is shown. In panel D or F, pentons are labeled with the number 5 and hexons with the number 6.

FIG 9

Coimmunoprecipitation of pU_L17, pU_L25, and pU_L31 in cells infected with wild-type and U_L25 mutant viruses. CV1 cells were infected with HSV-1(F) or the U_L25 mutants indicated above the panel. At 18 h postinfection, the cells were lysed and reacted with anti-pU_L17 or anti-pU_L31 antibodies. Antigen-antibody complexes were purified and electrophoretically separated on a denaturing polyacrylamide gel. (A) Viruses used for infection are indicated at the top of each lane. The first 3 lanes contain cellular lysates only. Lanes 4 to 8 contain immunoprecipitation reactions. Labels above each lane indicate the virus used for infection, followed by a forward slash and the antibody used for immunoprecipitation. To the right are the antigens recognized by immunoblotting. (B) The cell lysates (lanes 1 to 4) and materials immunoprecipitated with pU_L17-specific antibody (lanes 5 to 8) were transferred to nitrocellulose and subsequently probed with the antibodies indicated to the right. Bound antibodies were visualized using appropriate conjugates and ECL chemistry.