Nuclei move to specific locations to polarize migrating and differentiating cells. Many nuclear movements are microtubule-dependent. However, nuclear movement to reorient the centrosome in migrating fibroblasts occurs through an unknown actin-dependent mechanism. Here, we found that linear arrays of outer (nesprin2G) and inner (SUN2) nuclear membrane proteins assembled on and moved with retrogradely moving dorsal actin cables during nuclear movement in polarizing fibroblasts. Inhibition of nesprin2G, SUN2 or actin prevented nuclear movement and centrosome reorientation. The coupling of actin cables to the nuclear membrane for nuclear movement via specific membrane proteins indicates that like plasma membrane integrins, nuclear membrane proteins assemble into actin-dependent arrays for force transduction.

The capsids of neurotropic herpesviruses have the remarkable ability to move in specific directions within axons. By modulating bidirectional capsid transport to favor either retrograde (minusend) or anterograde (plus-end) motion, these viruses travel to sensory ganglia or peripheral tissue at specific stages of infection. By using correlative motion analysis to simultaneously monitor the trafficking of distinct viral proteins in living neurons, we demonstrate that viral ''tegument'' proteins are complexed to capsids moving in axons. The removal of a subset of tegument proteins from capsids invariably preceded retrograde transport to the cell body in sensory ganglia, whereas addition of these proteins was coupled to anterograde transport of progeny capsids to the distal axon. Although capsid transport never occurred without associated tegument proteins, anterograde-specific tegument proteins were competent to travel to the distal axon independent of capsids. These findings are compatible with a model of viral bidirectional transport in which tegument proteins direct capsid traffic to specific intracellular locations during the infectious cycle.neuron ͉ virus

Mutations in LMNA, which encodes A-type lamins, result in disparate diseases, known collectively as laminopathies, that affect distinct tissues, including striated muscle and adipose tissue. Lamins provide structural support for the nucleus and sites of attachment for chromatin, and defects in these functions may contribute to disease pathogenesis. Recent studies suggest that A-type lamins may facilitate connections between the nucleus and the cytoskeleton mediated by nuclear envelope nesprin and SUN proteins. In mammalian cells, however, interfering with A-type lamins does not affect the localization of these proteins. Here, we used centrosome orientation in fibroblasts, which requires separate nuclear and centrosome positioning pathways, as a model system to understand how LMNA mutations affect nucleus-cytoskeletal connections. We find that LMNA mutations causing striated muscle diseases block actin-dependent nuclear movement, whereas most that affect adipose tissue inhibit microtubule-dependent centrosome positioning. Genetic deletion or transient depletion of A-type lamins also blocked nuclear movement, showing that mutations affecting muscle exhibit the null phenotype. Lack of A-type lamins, or expression of variants that cause striated muscle disease, did not affect assembly of nesprin-2G and SUN2 into transmembrane actinassociated nuclear (TAN) lines that attach the nucleus to retrogradely moving actin cables. Nesprin-2G TAN lines were less stable, however, and slipped over the nucleus rather than moving with it, indicating that they were not anchored. Nesprin-2G TAN lines also slipped in SUN2-depleted cells. Our results establish A-type lamins as anchors for nesprin-2G-SUN2 TAN lines to allow productive movement and proper positioning of the nucleus by actin.nesprin SUN | linker of nucleoskeleton and cytoskeleton complex | muscular dystrophy L amin A and lamin C, the predominant A-type lamins, are expressed in most differentiated somatic cells. Yet, different mutations in the LMNA gene encoding these proteins result in a variety of diseases that affect specific tissues. LMNA mutations cause autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) and related diseases with dilated cardiomyopathy (DCM) that affect cardiac muscle and skeletal muscle to variable degrees (1). Other LMNA mutations cause Dunnigan-type familial partial lipodystrophy (FPLD) that affects adipose tissue (2), Charcot-Marie-Tooth type 2B1 disease that affects peripheral neurons (3), and progeroid syndromes with features of accelerated aging (4, 5). The mechanism whereby mutations in a single gene that is widely expressed cause such diverse diseases remains a puzzle.There are two prevailing hypotheses to explain the pathogenesis of laminopathies. The mechanical stress hypothesis proposes that alterations in A-type lamins compromise nuclear integrity in tissues susceptible to stress, such as striated muscle. This model is supported by findings that A-type lamin deficiency disrupts nuclear integrity in model systems (6, 7). The secon...

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.Adeno-associated viruses (AAV) are members of the dependent parvovirus family that requires helper viruses, such as adenovirus, to initiate productive infection and genome replication (27). Six distinct serotypes of primate AAV have been reported to date (2,5,6,26,31,41). Cloning and sequence characterization of these serotypes indicate that they share a similar genomic organization, which consists of two large open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The ITR structure is the minimal sequence required for AAV DNA replication, provirus integration, and packaging of progeny AAV DNA into virus particles. The left ORF encodes four nonstructural Rep proteins. These proteins not only are the regulators of AAV transcription (22) but also are involved in AAV replication (35) and virus assembly (21) and play a role in site-specific integration of the viral genome into the host chromosome during latent infection (1, 24). The sequences of the Rep ORFs of AAV-2, AAV-3, AAV-4, and AAV-6 are approximately 85% identical, but AAV-5 has only 54.5% homology with the other AAV serotypes (5). The right half of the AAV genome encodes three viral capsid proteins referred to as VP1, VP2, and VP3 and is less conserved than the Rep ORF. Although AAV-2, AAV-3, and AAV-6 share about 80% h...

Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.Viruses must overcome the diffusion barrier of the cytoplasm to effectively replicate in mammalian cells. This is most dramatically exemplified with neurotropic infections, such as those of the alphaherpesviruses, during which virus particles may translocate several centimeters or more between axon terminals and neuronal cell bodies. Intracellular transport of alphaherpesvirus particles to the nucleus in both neurons and non-neuronal cells is dependent on microtubules (20,24,27,40).The alphaherpesvirus virion is composed of four structural elements. The viral genome consists of a linear doublestranded DNA (ca. 120 to 230 kbp) that is housed within a proteinaceous capsid having icosahedral symmetry (ϳ120-nm diameter). The capsid is enclosed within a host-derived lipid envelope, and between the capsid and the envelope is a collection of viral proteins collectively referred to as the tegument. Upon entry into a cell the viral envelope fuses with the cellular plasma or endosomal membrane, depositing the capsid and tegument into the cytosol (10,28,29). At this phase, many tegument proteins are removed from the capsid. However, at least three tegument proteins (VP1/2, UL37, and US3) remain associated with capsids as they travel toward the nucleus (12, 23). After replication and assembly of capsids in the nucleus, progeny capsids translocate to the cytosol where they are again found associated with the VP1/2, UL37, and US3 tegument proteins (8, 13). These capsid/tegument complexes ultimately bud into a component of the secretory pathway and egress from the cell (reviewed in reference 26). The dynamics of capsid transport and assembly in the cytoplasm are poorly understood.Although many alphaherpesvirus proteins can interact with cellular microtubule-based motors, no herpesvirus proteins are currently known to be required for capsid transport (3,4,6,19,25,31,47). The presence of VP1/2, UL37, and US3 on cytosolic capsids makes them prime candidates as effectors of intracellular capsid transport. Of particular interest to the present study, cells infected with viruses lacking either VP1/2 or UL37 assemble genome-containing capsids in the nucleus, and these capsids egress to the cytoplasm similar to capsids of wild-type viruses. However, once in the cytoplasm, unenveloped capsids lacking either VP1/2 or UL37 accumulate, and re-envelopment is rare or nonexistent (1, 2, 9, 16, 17...