Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.     

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic?dextran polymers. Thus, kinesin-1 uncoats viral DNA?and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.     

Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation

BACKGROUND: The cytoskeleton and associated motors play an important role in the establishment of intracellular polarity. Microtubule-based transport is required in many cell types for the asymmetric localization of mRNAs and organelles. A striking example is the Drosophila oocyte, where microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. RESULTS: Here, we show that mRNA localization and nuclear positioning at mid-oogenesis depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. CONCLUSION: The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.     

Actomyosin Pulls to Advance the Nucleus in a Migrating Tissue Cell

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.     

Actomyosin Pulls to Advance the Nucleus in a Migrating Tissue Cell

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.     

Yaba virus-specific DNA in the host cell nucleus.

DNA-DNA hybridization studies show that Yaba virus-specific DNA is present in the host cell nucleus late in the infection cycle. The nuclear DNA appears to exist as a complete genome, not convalently linked to host cell DNA, as demonstrated by sedimentation analyses. The DNA apperas to be synthesized in the nucleus, since its level of incorporation of label is ten times the background incorporation detectable in the cytoplasm. Extraction of the nuclei by treatment with SDS and EDTA after precipitation with 1 M NaCl separates most of cellular DNA from the Yaba virus-specific DNA.     

Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.     

Agrobacterium VirE2 gets the VIP1 treatment in plant nuclear import.

Agrobacterium tumefaciens transforms plant cells by targeting a large single-stranded DNA molecule (T-strand) to the plant nucleus. The host cell contribution to nuclear import and transformation is the focus of several current articles. Recently, plant proteins have been identified that promote nuclear import of the T-strand. In particular, VIP1 might couple transformation to the importin-dependent nuclear import pathway and deliver the T-strand to chromatin, thereby promoting integration into the host genome.     

Histone H3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus

Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata.     

Kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus

Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro. HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the alpha3beta1 integrin as one of its entry receptors. Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P. P. Naranatt, S. M. Akula, C. A. Zien, H. H. Krishnan, and B. Chandran, J. Virol. 77:1524-1539, 2003). However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined. Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways. The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection. In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection. Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases. The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus. In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA. Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules. The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA. HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases. These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection. This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus. These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.     

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.     

Motors in fungal morphogenesis: cooperation versus competition

Fungal tip growth underlies substrate invasion and is essential for fungal virulence. It requires the activity of molecular motors that deliver secretory vesicles to the growth region or which mediate bi-directional motility of early endosomes. Visualizing motors and their cargo in living fungal cells revealed unexpected cooperation between motors in membrane trafficking: (1) Class V chitin synthase, which has a class 17 myosin motor domain, moves bi-directionally, with myosin-5 and kinesin-1 cooperating in delivery to the growth region, and dynein taking it back to the cell centre. The myosin-17 motor domain competes with dynein by tethering the chitin synthase to the plasma membrane before exocytosis; (2) Long-range endosome motility is based on a cooperation of kinesin-3 and dynein, but towards the microtubule plus-end dynein competes with kinesin-3 to prevent the organelles from 'falling off the track'. These results reveal a fine-balanced network of cooperative and competitive motor activity, required for fungal morphogenesis.     

Microtubule motors in eukaryotic spindle assembly and maintenance

The spindle is a microtubule-based structure that facilitates chromosome segregation during mitosis and meiosis. Spindle assembly from dynamic microtubule building blocks is a major challenge for the dividing cell and a process that critically requires microtubule motors. In this review we focus on the mechanisms by which microtubule motors shape the spindle. Specifically, we address how motors are thought to move and arrange microtubules to form the characteristic bipolar morphology shared by all eukaryotic spindles as well as motor-dependent mechanisms of microtubule length regulation.     

Viral and host control of cytomegalovirus maturation

Maturation in herpesviruses initiates in the nucleus of the infected cell, with encapsidation of viral DNA to form nucleocapsids, and concludes with envelopment in the cytoplasm to form infectious virions that egress the cell. The entire process of virus maturation is orchestrated by protein-protein interactions and enzymatic activities of viral and host origin. Viral tegument proteins play important roles in maintaining the structural stability of capsids and directing the acquisition of virus envelope. Envelopment occurs at modified host membranes and exploits host vesicular trafficking. In this review, we summarize current knowledge of and concepts in human cytomegalovirus (HCMV) maturation and their parallels in other herpesviruses, with an emphasis on viral and host factors that regulate this process.     

Kinesins and microtubules: their structures and motor mechanisms

Atomic resolution three-dimensional structures of two oppositely directed kinesin motors - conventional kinesin and non-claret disjunctional (ncd) protein - are now available in their functional dimeric form. A detailed model of the microtubule has also been recently obtained by docking the 3.7 A structure of tubulin into a 20 A map of the microtubule. Recent structural studies of kinesin motors and their microtubule tracks are contributing to our current understanding of kinesin motor mechanisms.     

Association of adenovirus with the microtubule organizing center

Adenoviruses (Ad) must deliver their genomes to the nucleus of the target cell to initiate an infection. Following entry into the cell and escape from the endosome, Ad traffics along the microtubule cytoskeleton toward the nucleus. In the final step in Ad trafficking, Ad must leave the microtubule and establish an association with the nuclear envelope. We hypothesized that in cells lacking a nucleus, the capsid moves to and associates with the microtubule organizing center (MTOC). To test this hypothesis, we established an experimental system to examine Ad trafficking in enucleated cells compared to Ad trafficking in intact, mock-enucleated cells. Enucleation of a monolayer of A549 human lung epithelial cells was accomplished by depolymerization of the actin cytoskeleton followed by centrifugation. Upon infection of enucleated cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a discrete, centrally located site which colocalized with pericentrin, a component of the MTOC. MTOC-associated Ad had escaped from endosomes and thus had direct access to MTOC components. Ad localization at this site was sensitive to the microtubule-depolymerizing agent nocodazole, but not to the microfilament-depolymerizing agent cytochalasin B, indicating that intact microtubules were required to maintain the localization with the MTOC. Ad localization to the MTOC in the enucleated cells was stable, as demonstrated by continuing Ad localization with pericentrin for more than 5 h after infection, a strong preference for Ad arrival at rather than Ad departure from the MTOC, and minimal redistribution of Ad between MTOCs within a single cell. In summary, the data demonstrate that the Ad capsid establishes a stable interaction with the MTOC when a nucleus is not present, suggesting that dissociation of Ad from microtubules likely requires nuclear factors.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.