Plus-end tracking proteins, CLASPs, and a viral Akt mimic regulate herpesvirus-induced stable microtubule formation and virus spread

Abstract

Although microtubules (MTs) frequently form highly dynamic networks, subsets of MTs become stabilized in response to environmental cues and function as specialized tracks for vesicle and macromolecular trafficking. MT stabilization is controlled by specialized plus-end tracking proteins (+TIPs) whose accumulation at the MT ends is facilitated by the end-binding protein, EB1, and regulated by various signaling pathways. As cargoes themselves, viruses are dependent on MTs for their intracellular movement. Although many viruses affect MT organization, the potential contribution of MT stabilization by +TIPs to infection remains unknown. Here we show that early in infection of primary human fibroblasts, herpes simplex virus type 1 (HSV-1) disrupts the centrosome, the primary MT organizing center in many cell types. As infection progresses HSV-1 induces the formation of stable MT subsets through inactivation of glycogen synthase kinase 3beta by the viral Ser/Thr kinase, Us3. Stable MT formation is reduced in cells infected with Us3 mutants and those stable MTs that form cluster around the trans-Golgi network. Downstream of glycogen synthase kinase 3beta, cytoplasmic linker-associated proteins (CLASPs), specialized host +TIPs that control MT formation at the trans-Golgi network and cortical capture, are specifically required for virus-induced MT stabilization and HSV-1 spread. Our findings demonstrate the biological importance of +TIPs to viral infection and suggest that HSV-1 has evolved to exploit the trans-Golgi network as an alternate MT organizing center to facilitate virus spread.

Fig. 1.

HSV-1 induces MT stabilization in primary human fibroblasts. (A–C) NHDFs were mock-infected or infected with HSV-1 strain F at m.o.i. 20. (A) Samples were fixed at the indicated times and stained for Tyr-MTs (red) and Ac-MTs (green). Arrows highlight representative perinuclear dots of Tyr-tubulin at the centrosome. (B and C) Infected samples fixed at 9 h.p.i. were stained for (B) Tyr-MTs (red) and EB1 (green) or (C) Ac-MTs (green) and EB1 (red). Magnification 100×. Arrows point to Tyr-MT ends with EB1 comets (B) or Ac-MTs lacking EB1 (C). (D) NHDFs or HeLa cells were infected at m.o.i. 2 for 16 h with the indicated HSV-1 strains, and lysates were analyzed by WB using the indicated antibodies. Ac-Tub, acetylated tubulin; T-Tub, total tubulin. The arrow points to GFP-tagged Us11 in Patton strain HSV-1-GFP-Us11 (Patt-Us11).

Fig. 2.

Host Dia1 and Akt are not required for MT stabilization by HSV-1. (A) NHDFs were treated with Ctrl or Dia1 siRNAs and then mock-infected or infected at m.o.i. 10 for 16 h. (Left) Fixed samples were stained for Ac-MTs. (Right) Parallel samples were lysed and analyzed by WB with the indicated antibodies. (B–D) Dia1 has minimal effects on HSV-1 spread. NHDFs were treated with Ctrl or either of two different Dia1 siRNAs and then infected at m.o.i. 0.0005 for 3 d. (B) Representative phase and fluorescence (GFP) images of plaques formed by HSV-1-GFP-Us11. (C) WB analysis of lysates from B, using the indicated antibodies. HSV-1, antibody against HSV-1 virions. (D) Levels of infectious virus present in culture supernatants after infection with HSV-1 strain F, determined by titration on Vero cells. (E) NHDFs were treated with DMSO (−) or AKTVIII (+) and then infected at m.o.i. 10 for 16 h. (E, Top) Fixed samples were stained for Tyr-MTs (red) and Ac-MTs (green). (E, Bottom) WB analysis of lysates, using the indicated antibodies.

Fig. 3.

The kinase activity of Us3 induces stable MT formation. (A and B) NHDFs were mock-infected or infected at m.o.i. 10 for 9 h with the indicated viruses. (A) Fixed samples were stained for Tyr-MTs (red) and Ac-MTs (green). (B) Whole-cell extracts were analyzed by WB with the indicated antibodies. (C) U20S or HeLa cells were transfected with empty vector or plasmids encoding Flag-tagged Us3 or kinase inactive Us3 (K220A). Forty-eight hours later, lysates were analyzed by WB, using the indicated antibodies. WT Us3 autophosphorylates, resulting in altered migration compared with K220A. Ac-Tub, acetylated tubulin; T-Tub, total tubulin.

Fig. 4.

The TGN acts as an alternate MTOC in HSV-1-infected cells. NHDFs were mock-infected or infected with HSV-1 at m.o.i. 5 for 14 h. Cultures were treated for 3 h with DMSO or 10 μM nocodazole. Nocodazole was then washed out (w/o) for the indicated times. (A) Fixed samples were stained for Tyr-MTs (red) and TGN46 (green). (B) Higher-magnification images of samples from A illustrate Tyr-MT growth from TGN structures, highlighted by arrows. (C) Fixed samples were stained for TGN46 (red) and Ac-MTs (green). (D) Higher magnification of samples 15 min after w/o in B. Arrows point to Ac-MTs radiating from TGN46-positive structures.

Fig. 5.

CLASPs are required for HSV-1-induced stable MT formation and virus spread. NHDFs were treated with Ctrl or CLASP2 siRNAs and then infected. (A) Samples infected at m.o.i. 10 for 16 h were fixed and stained for TGN46 (red) and Ac-MTs (green). (B) Samples were infected at m.o.i. 5 for 16 h, and then levels of cell-associated (Cell) and supernatant (Sup) infectious virus were determined by titration on Vero cells. (C) siRNA-treated NHDFs were infected with HSV-1-GFP-Us11 at m.o.i. 0.0005 for 3 d. (Left) Representative phase and fluorescence (GFP) images of plaques formed in control or CLASP2 siRNA-treated cultures. (Right) WB analysis of whole-cell extracts shows depletion of CLASP1, CLASP2, and CRMP2 and their effects on the accumulation of early (ICP22), late (Us11), and HSV-1 virion (HSV-1) proteins.