Role of Cellular Actin in the Gene Expression and Morphogenesis of Human Respiratory Syncytial Virus

Abstract

Cytoskeletal protein actin and nonactin cellular proteins were essential for human respiratory syncytial virus (RSV) gene expression. In vitro, specific antibodies against actin inhibited RSV transcription, whereas antibodies against other cytoskeletal proteins had little or no effect. Affinity purified cellular actin or bacterially expressed recombinant actin activated RSV transcription. However, optimal transcription required additional cellular protein(s) that appeared to function as accessory factor(s) for actin. In the absence of actin, these proteins did not activate viral transcription. Purified viral nucleocapsids contained actin, but no cytokeratin, tubulin, or vimentin. Cytochalasin D or DNasel--agents that destabilize actin polymers--had little effect on RSV transcription. RSV infection itself seemed to alter the structure of the cellular actin filaments. Treatment of infected cells with cytochalasin D produced a more severe disruption of the filaments and drastically reduced the production of infectious virus particles but still had little effect on intracellular synthesis of viral macromolecules. Thus actin seems to serve a dual role in RSV life cycle: its monomeric form as well as polymeric form activate viral transcription, while only the microfilament form may take part in viral morphogenesis and/or budding.

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

“…Several cytoskeletal proteins, including actin isoforms, cofilin, and filamin-1, were detected by mass spectrometry analyses of purified viral filaments (although purity was difficult to assess due to the cell-associated nature of filaments). There is ample evidence of a role for polymerized actin and actinregulatory proteins in assembly (4,6,11,22,30,32,49,53,59). However, the role of polymerized actin seems to be limited to the actual budding/release event (11,30,32), which was shown previously to occur at sites of lipid rafts (8,9,22,27,31,36,42,49,50).…”

Ectopic expression of miR-126*, an intronic product of the vascular endothelial EGF-like 7 gene, regulates prostein translation and invasiveness of prostate cancer LNCaP cells

“…Protein was measured by immunoblot (Western blot) using the horseradish peroxidaseconjugated secondary antibody and the ECL detection system (Pierce) as described previously [39]. Rabbit antihuman prostein antibody and goat antihuman Egfl7 antibody were from Abcam and R&D Systems, respectively.…”

Protein-Protein Interactions in RSV Assembly: Potential Targets for Attenuating RSV Strains

“…Studies using in vitro reconstituted nucleocapsid, mini-genome systems or nucleocapsids purified from RSV-infected cells have shown that optimal transcriptional activity of the nucleocapsid also requires cellular proteins [31,32]. Subsequent studies identified actin as one of the specific cellular factors required [31]; nucleocapsids purified from RSV infected cells always contain some actin [14].…”

“…Subsequent studies identified actin as one of the specific cellular factors required [31]; nucleocapsids purified from RSV infected cells always contain some actin [14]. The role of the actin-binding protein profilin in nucleocapsid activity is less clear; although it enhances transcriptase activity in cellfree systems [33], profilin knockdown in cell lines has no effect on RSV replication or transcriptase activity [34].…”

“…Clearly, trafficking of M between the nuclear and cytoplasmic compartments is a key aspect of its multiple functions in infected cells/viral infection. [11,12,30,31]. Thus, actin may be involved in RSV budding as well facilitating movement of nucleocapsid and envelope glycoproteins to the cell surface.…”