Cellular and Viral Determinants of HSV-1 Entry and Intracellular Transport towards Nucleus of Infected Cells

Abstract

HSV-1 employs cellular motor proteins and modulates kinase pathways to facilitate intracellular virion capsid transport. Previously, we and others have shown that the Akt inhibitor miltefosine inhibited virus entry. Herein, we show that the protein kinase C inhibitors staurosporine (STS) and gouml inhibited HSV-1 entry into Vero cells, and that miltefosine prevents HSV-1 capsid transport toward the nucleus. We have reported that the HSV-1 UL37 tegument protein interacts with the dynein motor complex during virus entry and virion egress, while others have shown that the UL37/UL36 protein complex binds dynein and kinesin causing a saltatory movement of capsids in neuronal axons. Co-immoprecipitation experiments confirmed previous findings from our laboratory that the UL37 protein interacted with the dynein intermediate chain (DIC) at early times post infection. This UL37-DIC interaction was concurrent with DIC phosphorylation in infected, but not mock-infected cells. Miltefosine inhibited dynein phosphorylation when added before, but not after virus entry. Inhibition of motor accessory protein dynactins (DCTN2, DCTN3), the adaptor proteins EB1 and the Bicaudal D homolog 2 (BICD2) expression using lentiviruses expressing specific shRNAs, inhibited intracellular transport of virion capsids toward the nucleus of human neuroblastoma (SK-N-SH) cells. Co-immunoprecipitation experiments revealed that the major capsid protein Vp5 interacted with dynactins (DCTN1/p150 and DCTN4/p62) and the end-binding protein (EB1) at early times post infection. These results show that Akt and kinase C are involved in virus entry and intracellular transport of virion capsids, but not in dynein activation via phosphorylation. Importantly, both the UL37 and Vp5 viral proteins are involved in dynein-dependent transport of virion capsids to the nuclei of infected cells.ImportanceHerpes simplex virus type-1 enter either via fusion at the plasma membranes or endocytosis depositing the virion capsids into the cytoplasm of infected cells. The viral capsids utilize the dynein motor complex to move toward the nuclei of infected cells using the microtubular network. This work shows that inhibitors of the Akt kinase and kinase C inhibit not only viral entry into cells but also virion capsid transport toward the nucleus. In addition, the work reveals that the virion protein ICP5 (VP5) interacts with the dynein cofactor dynactin, while the UL37 protein interacts with the dynein intermediate chain (DIC). Importantly, neither Akt nor Kinase C was found to be responsible for phosphorylation/activation of dynein indicating that other cellular or viral kinases may be involved.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1

“…Dynein must be phosphorylated to obtain an active conformation ( 54 , 55 , 59 – 61 ). Recently, it was shown that upon infection of epithelial cells with HSV-1, dynein intermediate chain 1B was phosphorylated at position S80 in an Akt- and protein kinase C (PKC)-independent manner ( 62 ). It was speculated that US3’s kinase function could mediate the activation of dynein throughout the retrograde transport process.…”

A pseudorabies virus serine/threonine kinase, US3, promotes retrograde transport in axons via Akt/mToRC1

“…Recently, it was shown that upon infection of epithelial cells with HSV-1, dynein intermediate chain 1B was phosphorylated at position S80 in an Akt and PKC independent manner 58 . We have also observed dynein phosphorylation (at position T88) during PRV infection that was US3 dependent (A. D. Esteves and L. W. Enquist, unpublished data).…”

Small molecule RAF265 as an antiviral therapy acts against HSV‐1 by regulating cytoskeleton rearrangement and cellular translation machinery

“…34,35 Major capsid protein ICP5 is involved in the transport of virion capsids to the nuclei. 35 During the late stage of infection, ICP5 localizes in DNA replication compartments, sites of infected cell nuclei, in which newly replicated viral DNAs insert into preformed capsids. 35,36 In the current study, fluorescence microscopy was utilized to observe HSV-1-ICP5 antibody staining positive cells; it seems that RAF265 possibly facilitates the viral entry process (Figure 2D).…”