U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

Abstract

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

FIG. 1

Digitized scanned image of immunoblots probed with antisera directed against U_L34 and U_L31 proteins. (A) Vero cells were either mock infected (lane 1) or infected for 18 h with HSV-1(F) (lane 2) or HSV-2(G) (lane 3). Cellular lysates were prepared, separated on an SDS-polyacrylamide gel, blotted to nitrocellulose, and probed with anti-U_L34 antibody. (B) HEp-2 cells were mock infected (lane 1), infected with wild-type HSV-1 (F) virus (lane 2), or infected with U_L31 null mutant virus (lane 3). Lysates were prepared at 16 h.p.i., subjected to electrophoresis on an SDS–10% polyacrylamide gel, blotted to nitrocellulose, and probed with antiserum raised against a purified U_L31-GST fusion protein. Sizes of protein standards are indicated between panels A and B.

FIG. 2

Digitized confocal optical sections of a time course of U_L34 protein localization. HEp-2 cells were mock infected (A and B), infected at an MOI of 10 with U_L34 null virus (vRR1072) for 10 h (C), or infected at an MOI of 10 with HSV-1(F) for 4 h (D to I), 6 h (J to L), 8 h (M to O), 10 h (P to R), or 20 h (S to X). All cells were fixed with formaldehyde, permeablized, and immunostained with chicken anti-U_L34 antibody, detected with donkey anti-chicken Ig-Texas Red conjugate either alone (C) or with mouse anti-nucleoporin p62 detected with goat anti-mouse Ig-FITC conjugate (A, B, and D to X). Arrows indicate positions of invaginations and projections of the nuclear envelope (B and P to R) or membranous nuclear peripheral structures (V to X). Original magnifications: X630 (A to U); X1000 (V to X).

FIG. 3

Digital confocal images of HEp-2 cells infected with wild-type HSV-1(F) at an MOI of 5. Cells were fixed and permeablized in ice-cold methanol at 0, 4, 8, 12, 16, and 20 h.p.i. Fixed cells were immunostained with U_L31 rabbit antiserum and ICP4 monoclonal antibody. U_L31-specific immunofluorescence was detected with a Texas Red-conjugated donkey anti-rabbit Ig antibody, and ICP4-specific immunofluorescence was detected with FITC-conjugated donkey anti-mouse Ig antibody. Original magnification, X600.

FIG. 4

Digital confocal image of HEp-2 cells infected with various viruses and fixed in methanol at 16 h.p.i. Using an MOI of 5, HEp-2 cells were infected with wild-type HSV-1 (WT), U_L34 null mutant virus vRR1072(tk⁺) (34−), repaired U_L34 mutant virus vRR1072Rep (34R), U_L31 null mutant virus R5132 (31−), or a repaired U_L31 mutant virus (R5132Rep) (U_L31R). Cells were stained with U_L31 protein-specific antiserum (green), U_L34 protein-specific antiserum (red), and ICP4-specific ascites fluid (blue). Bound immunoglobulins specific for U_L31 protein, U_L34, and ICP4 by reaction with FITC-conjugated donkey anti-rabbit Ig, Texas Red-conjugated donkey anti-chicken Ig, and Cy5-conjugated goat anti-mouse Ig antibody, respectively. Cells were analyzed by confocal laser scanning microscopy by acquiring images through the middle of the cell. For illustrative purposes, Cy5 staining was pseudocolored blue in the figure. Areas of U_L31 and U_L34 staining colocalization are indicated by a yellow color upon merging of the separate images (Merge). Original magnification, X600.

FIG. 5

Digital confocal images of transfected HEp-2 cells fixed and permeabilized with ice-cold methanol at 18 h posttransfection. (A) Cells were transfected with U_L31/pcDNA3 and probed with anti-U_L31-GST antibody. (B) Nomarski DIC image of same microscopic field as in panel A. (C) Merge of images in A and B. (D) HEp-2 cells were transfected with U_L34/pcDNA3 and stained with antiserum directed against U_L34 protein. (E) Nomarski DIC image of same field as in panel D. (F) Merge of images in D and E. (G) Cells were cotransfected with U_L31 and U_L34 constructs and costained with U_L31-specific and U_L34-specific antisera. U_L31-specific immunofluorescence is shown in panel G. (H) U_L34-specific immunofluorescence in same cotransfected cells as in panel H. (I) Merge of U_L31- and U_L34-specific immunofluorescence, showing colocalization of transiently expressed U_L31 and U_L34 proteins as visualized by a yellow color upon merging of the separate images. (J) Nomarski DIC image of cotransfected cell shown in panels G to K. (K) Nomarski DIC image merged with the U_L31- and U_L34-specific immunofluorescence. Original magnification, X600.

FIG. 6

Digitized fluorographic image of GST pull-down reactions using equal amounts of GST or GST fusion proteins reacted with either in vitro-expressed radiolabeled U_L31 protein (left panel) or radiolabeled U_L34 protein (right panel). GST pull-down reactions in lanes 2, 3, 5, and 6 were performed similarly. Radiolabeled protein was added to Sepharose beads bearing either GST or GST fused to the putative interaction partner. After incubation, the beads were washed extensively, followed by elution in SDS sample buffer and electrophoretic separation on a denaturing polyacrylmide gel. The gel was subjected to analysis on a Molecular Dynamics PhosphorImager and fluorography. Lane 1, product of U_L31 transcribed and translated in vitro. The amount of radiolabeled U_L31 protein loaded in lane 1 was approximately 20% of that used in the reactions shown in lanes 2 and 3. Lane 2, radiolabeled U_L31 protein bound to GST immobolized on Sepharose beads. Lane 3, radiolabeled U_L31 protein bound to Sepharose beads containing U_L34-GST. Lane 4, radiolabeled U_L34 protein produced in an in vitro transcription-translation reaction. The amount of U_L34 protein loaded in lane 4 was approximately 20% of that used in reactions shown in lanes 5 and 6. Lane 5, radiolabeled U_L34 protein bound to immobilized GST. Lane 6, radiolabeled U_L34 protein bound to immobilized U_L31-GST. Molecular weights are indicated in thousands to the left of the figure.

FIG. 7

Digital confocal images showing localization of U_L34 protein in cells infected with a U_S3 null virus. (A to F and J to R) HEp-2 cells were infected at an MOI of 10 with U_S3 null HSV-1(F) (R7039) for 6 h (A to C), 8 h (D to F), 10 h (J to L), and 20 h (M to R). Infected cells were fixed with formaldehyde and immunostained with chicken anti-U_L34 antibody detected with donkey anti-chicken Ig-Texas Red conjugate and with mouse anti-nucleoporin p62 detected with goat anti-mouse Ig-FITC conjugate. (G to I) HEp-2 cells were transfected with an LBR-GFP expression construct and allowed to express for 24 h. Transfected cells were subsequently infected for 8 h with U_S3 null virus R7039, fixed with formaldehyde, and immunostained with chicken anti-U_L34 detected with donkey anti-chicken Ig-Texas Red conjugate. (S to X) HEp-2 cells were infected with HSV-1(F) (S to U) or R7039 (V to X) at an MOI of 10 for 10 h fixed, permeabilized, and immunostained with anti-U_L34 and anti-nucleoporin p62. Confocal Z-sections are shown in panels A to R, and confocal Z-stacks are shown in panels S to X. Arrows in panels A through F indicate positions of perinuclear U_L34-nucleoporin p62 structures. Original magnifications: A to O and S to X, X630; P to R, X1,000.

FIG. 8

Digital confocal images showing transiently tranfected HEp-2 cells. Cells were transfected with a U_L34 expression construct (A to C) or a U_S3 expression construct (D to F) or cotransfected with U_L34 and U_S3 expression constructs (G to I). At 24 h posttransfection cells were fixed with formaldehyde and immunostained with chicken anti-U_L34 antibody detected with donkey anti-chicken Ig-Texas Red conjugate and rabbit anti-U_S3 detected with goat anti-rabbit Ig-FITC conjugate. Shown are confocal Z-sections. The high level of background U_L34 staining seen in cells transfected with U_S3 alone (panels D to F) was not a regular feature of these experiments. Original magnification, X630.

FIG. 9

Digital confocal images of HEp-2 cells infected with U_S3 null mutant virus R7037 at an MOI of 5 that were fixed and permeabilized in ice-cold methanol at 16 h.p.i. Cells were immunostained with antisera directed against U_L31 and U_L34 proteins. U_L31 protein-specific binding (green) was detected with FITC-conjugated donkey anti-rabbit Ig antibody, and U_L34 protein-specific binding (red) was detected with Texas Red-conjugated donkey anti-chicken Ig antibody. Areas of colocalization of U_L31 and U_L34 proteins are indicated by a yellow color upon merging of the images. Confocal Z-sections were acquired in 0.4-μm slices through the entire thickness of the cell. Digital slices from the top (row labeled Top) of the cell or the middle of the cell (row labeled Middle) are shown. A confocal Z-stacked image derived from the Z-sections is shown in the row labeled Stacked. Original magnification, X600.