Vaccinia locomotion in host cells: Evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2

Zeile, William L.; Condit, Richard C.; Lewis, Jackie I.; Purich, Daniel L.; Southwick, Frederick S.

doi:10.1073/pnas.95.23.13917

Cited by 20 publications

(24 citation statements)

References 32 publications

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“…cell density), inhibition/activation of various protein kinase cascades, induction of cellular damage, or modification of the cytoskeleton have been unsuccessful (data not shown). However, here we show that one strategy for inducing nuclear accumulation of zyxin is to inactivate its nuclear export signal by deletion or by treating cells with the generalized nuclear export inhibitor leptomycin B. Interestingly, Zeile and colleagues have also reported nuclear accumulation of endogenous zyxin after infection of cells with a vaccinia virus (55). Whether this altered distribution results from specific inactivation of zyxin's nuclear export signal or enhancement of zyxin's import activity remains to be resolved.…”

Section: Discussionmentioning

confidence: 89%

Targeting of Zyxin to Sites of Actin Membrane Interaction and to the Nucleus

Nix¹,

Fradelizi²,

Bockholt³

et al. 2001

Journal of Biological Chemistry

143

130

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The localization of proteins to particular intracellular compartments often regulates their functions. Zyxin is a LIM protein found prominently at sites of cell adhesion, faintly in leading lamellipodia, and transiently in cell nuclei. Here we have performed a domain analysis to identify regions in zyxin that are responsible for targeting it to different subcellular locations. The N-terminal proline-rich region of zyxin, which harbors binding sites for ␣-actinin and members of the Ena/VASP family, concentrates in lamellipodial extensions and weakly in focal adhesions. The LIM region of zyxin displays robust targeting to focal adhesions. When overexpressed in cells, the LIM region of zyxin causes displacement of endogenous zyxin from focal adhesions. Upon mislocalization of full-length zyxin, at least one member of the Ena/VASP family is also displaced, and the organization of the actin cytoskeleton is perturbed. Zyxin also has the capacity to shuttle between the nucleus and focal adhesion sites. When nuclear export is inhibited, zyxin accumulates in cell nuclei. The nuclear accumulation of zyxin occurs asynchronously with approximately half of the cells exhibiting nuclear localization of zyxin within 2.3 h of initiating leptomycin B treatment. Our results provide insight into the functions of different zyxin domains.Cells change shape, migrate, proliferate, and differentiate in response to extracellular cues. Such sensitivity to environmental conditions is necessary for multiple processes including development, cell-mediated immunity, and organ regeneration (1, 2). Although much has been learned about a cell's capacity to recognize and respond to extracellular cues, in many cases we do not understand how engagement of a particular cell surface receptor triggers changes in cell behavior. One present challenge, for example, is to understand how engagement of integrin receptors for extracellular matrix can stimulate cell motility and changes in gene expression. In the past few years, several proteins that may contribute to these processes have been identified. For example, zyxin, a protein that is co-localized with integrins at sites of membrane-substratum adhesion, has properties that suggest it could participate in both localized actin polymerization and communication to the nuclear compartment.Zyxin is an elongate phosphoprotein composed of three C-terminal LIM domains, a proline-rich N-terminal region, and at least one nuclear export signal (NES) 1 (Fig. 1A) (3-6). Two binding partners for the LIM region of zyxin, H-warts/LATS1 and members of the cysteine-rich protein (CRP) family, have been identified (5, 7). H-warts/LATS1 is a tumor suppressor that is postulated to be an important regulator of mitotic progression (7). CRPs are thought to stabilize actin-rich structural elements in muscle (8,9). The N-terminal proline-rich region of zyxin displays docking sequences for several proteins implicated in actin assembly and organization including ␣-actinin and members of the Ena (enabled)/VASP (vasodilator-stimulate...

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Section: Discussionmentioning

confidence: 89%

Targeting of Zyxin to Sites of Actin Membrane Interaction and to the Nucleus

Nix¹,

Fradelizi²,

Bockholt³

et al. 2001

Journal of Biological Chemistry

143

130

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“…The use of a profilin-actin motility system has been implicated in the cell-cell spread of the bacterial pathogens Listeria monocytogenes (34) and Shigella flexneri (36). Vaccinia virus is known to use an actin-based motility system for cell-cell spread (10), and a role for profilin in the vaccinia virus-induced actin "rocket tails" has been suggested (37). Curiously, vaccinia virus encodes a viral homolog of profilin.…”

Section: Discussionmentioning

confidence: 99%

“…The profilin antibody was raised in rabbits against purified recombinant human profilin-1 (36,37) and was a generous gift from William Zeile and Frederick Southwick (University of Florida). Secondary antibodies conjugated to horseradish peroxidase were obtained from Sigma (St. Louis, Mo.…”

Section: Antibodiesmentioning

confidence: 99%

Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA

Burke

Mahoney

Almo

et al. 2000

J Virol

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Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-L-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-Lproline matrix, stimulated the actin-saturated RSV transcription by 2.5-to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-L-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.Human respiratory syncytial virus (RSV) is a major pathogen of the lower respiratory tracts of young infants (7). RSV belongs to the Pneumovirus genus within the Paramyxoviridae family. Like other members of this family, RSV has a nonsegmented, negative-strand RNA genome. The RSV genes and genome organization are unique among paramyxoviruses in many respects; the order of genes on the 15,222-kb RSV genomic RNA is 3Ј-(leader)-NS1-NS2-N-P-M-SH-G-F-M2-L-(trailer)-5Ј (14). The RSV nucleocapsid core consists of the viral genomic RNA wrapped with N protein (called the N-RNA template), the phosphoprotein P, the transcription elongation factor M2, and the major subunit of the RNA-dependent RNA polymerase, L (5, 9, 17, 21).As part of our ongoing investigation of the mechanisms of RSV gene expression, we have embarked on the characterization of the various components of the RSV RNA transcription machinery. Our initial studies showed that the viral nucleocapsid core alone was incapable of transcription in vitro; however, the addition of uninfected cell extract restored transcriptional activity (1). Subsequent fractionation of the cell extract revealed that cellular actin is both necessary and sufficient to reconstitute in vitro transcription (5). While that study constituted the first detailed report of a cytoskeletal protein acting as a bona fide transcription factor for RSV, it remained unknown whether actin-modulatory proteins played any role in the process. In the same study, however, we demonstrated that actin alone did not activate viral transcription to the same degree as the whole-cell lysate did. Thus, it was proposed that at least one ...

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“…After assembly, actin filaments are also cross-linked to form a dense rocket tail structure that subsequently undergoes pointed-end disassembly by cytoplasmic actin depolymerizing factors. Except for N-WASP and cdc42 replacing ActA in stimulating Arp2/3-mediated filament nucleation [Higgs and Pollard, 2001], the generality of this mechanism has been demonstrated in studies of Shigella , Rickettsia [Heinzen et al, 1999], vaccinia virus [Zeile et al, 1998] as well as such endogenous host-cell organelles as endosomes, lysosomes [Taunton et al, 2000], and pinosomes [Merrifield et al, 1999].…”

Section: Introductionmentioning

confidence: 96%

Actin‐based phagosome motility

Zhang

Southwick

Purich

2002

Cell Motil. Cytoskeleton

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Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation.

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Vaccinia locomotion in host cells: Evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2

Cited by 20 publications

References 32 publications

Targeting of Zyxin to Sites of Actin Membrane Interaction and to the Nucleus

Targeting of Zyxin to Sites of Actin Membrane Interaction and to the Nucleus

Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA

Actin‐based phagosome motility

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