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.M icrotubules (MTs) function in a variety of processes, including directed intracellular trafficking of cargos and changes in cell shape, polarity, and motility (1, 2). Consisting of polarized heteropolymers of α/β-tubulin, in most cells MT minusends are anchored at the perinuclear MT organizing center (MTOC), whereas their plus-ends radiate toward the cell periphery. In proliferating cells, the majority of MTs are highly dynamic, growing and shrinking through the addition or loss of tubulin subunits primarily at the plus-end. Dynamic instability facilitates intracellular sensing through "search-and-capture." On encountering targets such as the cell cortex and in response to specific signals, subsets of MTs become stabilized and acquire posttranslational modifications such as acetylation and detyrosination (3). Stable MTs are recognized by specific motor proteins and act as specialized tracks for vesicle transport (1, 3). MT stabilization is mediated by proteins that track dynamic MT plusends and influence rates of MT growth, pause, and collapse, known as plus-end tracking proteins (+TIPs) (4). Central to plusend tracking is EB1, a member of the end-binding family of proteins that recognizes growing MT ends. Although many +TIPs are capable of associating with MTs, it is their interaction with EB1 that mediates their specific accumulation at MT plus-ends (4). +TIP activity and interaction with EB1 responds to signals including Rho-Dia act...

Role of non-motile microtubule-associated proteins in virus trafficking

“…Recent work showed that adenovirus, African swine fever virus (ASFV), influenza A virus, reoviruses, HSV-1, HIV-1, and Ebola virus all induce MT stabilization, often observed with concomitant posttranslational modifications such as acetylation and detyrosination in host cells (90)(91)(92)(93)(94)(95)(96). Specifically, HSV-1 is thought to promote both its trafficking to the nucleus and its egress by multiple mechanisms, including the stabilization and hyper acetylation of MTs by the tegument protein VP22 (94) and viral kinase Us2 (97), binding of the molecular chaperone Hsp90 (98), disruption of the centrosomal nature of the MT-organizing center (MTOC) and its transfer to the trans-Golgi network (97,99,100). In this context, cytoplasmic linker-associated proteins (CLASPs), which are specialized + TIPs that control MT formation at the trans-Golgi network, were found to be specifically required for virus-induced MT stabilization and HSV-1 spread (97).…”

“…Specifically, HSV-1 is thought to promote both its trafficking to the nucleus and its egress by multiple mechanisms, including the stabilization and hyper acetylation of MTs by the tegument protein VP22 (94) and viral kinase Us2 (97), binding of the molecular chaperone Hsp90 (98), disruption of the centrosomal nature of the MT-organizing center (MTOC) and its transfer to the trans-Golgi network (97,99,100). In this context, cytoplasmic linker-associated proteins (CLASPs), which are specialized + TIPs that control MT formation at the trans-Golgi network, were found to be specifically required for virus-induced MT stabilization and HSV-1 spread (97). Furthermore, initial engagement of HSV-1 particles onto MTs involves a + TIP complex comprising EB1, CLIP-170, and dynactin-1 (DCTN1) to initiate retrograde transport in infected cells (86).…”

Microtubule Regulation and Function during Virus Infection

“…Once HSV-1 particles reach the nucleus and establish productive infection, punctate pericentrin staining is disrupted and MTs lose centrosomal focus (70,71). While disrupting centrosomal function, HSV-1 stabilizes MTs that nucleate at the TGN through the activity of Us3, a viral Akt mimic that regulates broad cytoskeletal rearrangements during infection (70,72).…”

“…While disrupting centrosomal function, HSV-1 stabilizes MTs that nucleate at the TGN through the activity of Us3, a viral Akt mimic that regulates broad cytoskeletal rearrangements during infection (70,72). In particular, formation of stable MT arrays occurs through Us3-mediated activation of cellular proteins called cytoplasmic linker-associated proteins (CLASPs) (70). CLASPs are specialized ϩTIPs that function in both MT nucleation at the Golgi apparatus and MT capture at the cell periphery.…”

“…However, it is tempting to speculate that dystonin contributes to MT nucleation or stabilization, as it is a ϩTIP with no known motor function. As outward kinesin motors exhibit a preference for acetylated or detyrosinated MTs, stabilizing MTs that originate from the site of envelope acquisition would ensure movement of new virus particles toward the cell surface (70). Moreover, stable MTs often form through capture at specific regions of the plasma membrane, and herpesvirus egress has been reported to occur at regions enriched for LL55␤, an established CLASP-interacting protein (76,77).…”