WDR60-mediated dynein-2 loading into cilia powers retrograde IFT and transition zone crossing

The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homolog (WDR-60) and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing the availability of the retrograde motor at the ciliary tip. Consistently, we show that less dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT and the NPHP module as a roadblock to dynein-2 passage through the transition zone.

Rotavirus viroplasm biogenesis involves microtubule-based dynein transport mediated by an interaction between NSP2 and dynein intermediate chain

Rotaviruses are the causative agents of severe and dehydrating gastroenteritis in children, piglets, and many other young animals. They replicate their genomes and assemble double-layered particles in cytoplasmic electron-dense inclusion bodies called ‘viroplasms’. The formation of viroplasms is reportedly associated with the stability of microtubules. Although material transport is an important function of microtubules, whether and how microtubule-based transport influences the formation of viroplasms is still unclear. Here, we demonstrate that the small viroplasms move and fuse in living cells. We show that microtubule-based dynein transport affects rotavirus infection, viroplasm formation, and the assembly of transient enveloped particles (TEPs) and triple-layered particles (TLPs). The dynein intermediate chain (DIC) is shown to localize in the viroplasm and to interact directly with non-structural protein 2 (NSP2), indicating that DIC is responsible for connecting the viroplasm to dynein. The WD40 repeat domain of DIC regulates the interaction between DIC and NSP2, and the knockdown of DIC inhibited rotaviral infection, viroplasm formation, and the assembly of TEPs and TLPs. Our findings show that rotavirus viroplasms hijack dynein transport for fusion events, required for maximal assembly of infectious viral progeny. This study provides novel insights into the intracellular transport of viroplasms, which is involved in their biogenesis. Importance Because the viroplasm is the viral factory for rotavirus replication, viroplasm formation undoubtedly determines the effective production of progeny rotavirus. Therefore, understanding the virus–host interactions involved in the biogenesis of the viroplasm is critical for the future development of prophylactic and therapeutic strategies. Previous studies have reported that the formation of viroplasms is associated with the stability of microtubules, whereas little is known about its specific mechanism. Here, we demonstrate that rotavirus viroplasm formation takes advantage of microtubule-based dynein transport mediated by an interaction between NSP2 and DIC. These findings provide new insight into the intracellular transport of viroplasms.

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

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. Importance. Herpes 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.

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

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.