Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase and regulates nuclear localization of viral envelopment factors UL34 and UL31

Abstract

UL13 and Us3 are protein kinases encoded by herpes simplex virus 1. We report here that Us3 is a physiological substrate for UL13 in infected cells, based on the following observations. (i) The electrophoretic mobility, in denaturing gels, of Us3 isoforms from Vero cells infected with wild-type virus was slower than that of isoforms from cells infected with a UL13 deletion mutant virus (DeltaUL13). After treatment with phosphatase, the electrophoretic mobility of the Us3 isoforms from cells infected with wild-type virus changed, with one isoform migrating as fast as one of the Us3 isoforms from DeltaUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of DeltaUL13 resembles that of a recombinant virus lacking the Us3 gene (DeltaUs3) with respect to localization of the viral envelopment factors UL34 and UL31, whose localization has been shown to be regulated by Us3. UL34 and UL31 are localized in a smooth pattern throughout the nuclei of cells infected with wild-type virus, whereas their localization in DeltaUL13- and DeltaUs3-infected cells appeared as nuclear punctate patterns. These results indicate that UL13 phosphorylates Us3 in infected cells and regulates UL34 and UL31 localization, either by phosphorylating Us3 or by a Us3-independent mechanism.

FIG. 1.

(A) Immunoblot of electrophoretically separated lysates from Vero cells mock infected (lane 1) or infected with HSV-1(F) (lane 2), R7356 (lane 3), or R7356Rep (lane 4). Infected cells were harvested at 12 h postinfection and analyzed by immunoblotting with polyclonal antibody to Us3. Wt, wild type. (B) Immunoblots of electrophoretically separated lysates from Vero cells infected with HSV-1(F) (lanes 1 and 2) and R7356 (lanes 3 and 4). The infected cells were harvested at 12 h postinfection, solubilized, mock treated (lanes 1 and 3) or treated with CIP (lanes 2 and 4), and immunoblotted with antibody to Us3. (C) Immunoblots of electrophoretically separated lysates from Vero cells infected with R7356Rep (lanes 1 and 2) and R7356 (lane 3). The infected cells were harvested at 12 h postinfection, solubilized, mock treated (lanes 1 and 3) or treated with CIP (lane 2), and immunoblotted with antibody to Us3.

FIG. 2.

(A) Schematic diagram of the genome structures of wild-type (Wt) virus HSV-1(F) and the location of the Us3 gene. Line 1, linear representation of the HSV-1(F) genome. The unique sequences are represented as unique long (U_L) and short (Us) domains, and the terminal repeats flanking them are shown as open rectangles with the designation above each repeat. Line 2, structure of the genome domain containing the Us2, Us3, and Us4 open reading frames. Line 3, structure of the Us3 open reading frame. The shaded areas represent subdomains I to VI, which are conserved in eukaryotic protein kinases (68). Line 4, the domains of the Us3 gene used in these studies to generate MBP-Us3 fusion proteins. (B) CBB-stained images of phosphorylated Us3. Purified MBP-Us3-P2 (lanes 1 and 2), MBP-Us3-P1 (lanes 3 and 4), and MBP-LacZ (lanes 5 and 6) incubated in kinase buffer containing [γ-³²P]ATP and purified GST-UL13 (lanes 1, 3, and 5) or GST-UL13K176M (lanes 2, 4, and 6), separated on a denaturing gel, and stained with CBB. Molecular masses (kDa) are shown on the left. (C) Autoradiograph of the gel in panel B. (D) Purified MBP-Us3-P1 incubated in kinase buffer containing [γ-³²P]ATP and purified GST-UL13 and then either mock treated (lane 1) or treated with λ-PPase (lane 2), separated on a denaturing gel, and stained with CBB. (E) Autoradiograph of the gel in panel D.

FIG. 3.

Autoradiographic images of Us3 immunoprecipitates subjected to in vitro kinase assay. (A) Vero cells were mock infected (lane 1) or infected with HSV-1(F) (lane 2), R7041 (lane 3), or R7356 (lane 4); harvested at 12 h postinfection; and immunoprecipitated with antibody to Us3. The immunoprecipitates were incubated in kinase buffer containing [γ-³²P]ATP, separated on a denaturing gel, transferred to a nitrocellulose membrane, and analyzed by autoradiography. (B) Immunoblot of the nitrocellulose membrane in panel A using anti-Us3 antibody. (C) Immunoprecipitates prepared as in panel A were either mock treated (lanes 1 and 3) or treated with λ-PPase (lanes 2 and 4), separated on a denaturing gel, transferred to a nitrocellulose membrane, and analzyed by autoradiography. Wt, wild type. (D) Immunoblot of the nitrocellulose membrane in panel C using anti-Us3 antibody.

FIG. 4.

(A) Immunoblot of electrophoretically separated lysates from Vero cells mock infected (lane 1) or infected with HSV-1(F) (lane 2), R7041 (lane 3), or R7356 (lane 4) at an MOI of 5. Infected cells were harvested at 12 h postinfection and immunoblotted with anti-UL31 antibody. (B) Caspase 3/7 activity of infected SK-N-SH cells after induction of apoptosis by osmotic shock. SK-N-SH cells were mock infected or infected with HSV-1(F), R7041, or R7356. At 12 h postinfection, the cells were exposed to sorbitol for 1 h, incubated for an additional 5 h, harvested, and assayed for caspase 3/7 activity using a Z-DEVD-aminoluciferin substrate. The values are the means and standard deviations for three independent experiments. Wt, wild type.

FIG. 5.

Digital confocal microscope images showing localization of UL34, UL31, and nucleoporin p62 proteins in Vero cells infected with HSV-1(F) (A, E, I, M, and Q), R7041 (B, F, J, N, and R), R7356 (C, G, K, O, and S), and R7356Rep (D, H, L, P, and T). At 12 h postinfection, infected cells were fixed, permeabilized, and immunostained with rabbit polyclonal antibody to UL34 (A to H) detected with FITC-conjugated anti-rabbit IgG antibody, rabbit polyclonal antibody to UL31 (I to P) detected with Alexa Fluor 488-conjugated anti-rabbit IgG antibody, or mouse monoclonal antibody to nucleoporin p62 (Q to T) detected with Alexa Fluor 488-conjugated anti-mouse IgG antibody.

FIG. 6.

(A) Digital confocal microscope images showing localization of UL34 in Vero cells infected with HSV-1(F) (a and d), R7041 (b and e), and R7356 (c and f). At 15 h postinfection, infected cells were fixed, permeabilized, and immunostained with rabbit polyclonal antibody to UL34 (a to c) detected with FITC-conjugated anti-rabbit IgG antibody or chicken polyclonal antibody to UL34 (d to f) detected with FITC-conjugated anti-chicken IgG antibody. (B) Digital confocal microscope images showing localization of UL34 and UL31 in Vero cells infected with HSV-1(F) (a, e, and i), R7041 (b, f, and j), R7356 (c, g, and k), and R7356Rep (d, h, and l). At 15 h postinfection, infected cells were fixed, permeabilized, and double labeled with a combination of chicken polyclonal antibody to UL34 (a to d) and rabbit polyclonal antibody to UL31 (e to h) and then detected with FITC-conjugated anti-chicken IgG antibody (green fluorescence) and Alexa-546-conjugated anti-rabbit IgG antibody (red fluorescence). Single-color images were captured separately and are shown in the upper (UL34) (a to d) and middle (UL31) (e to h) panels; the lower panels (i to l) represent simultaneous acquisitions of both colors. The yellow colors visualized in the merged images represent colocalization of UL34 and UL31.