The cascade of events that leads to vaccinia-induced actin polymerization requires Src-dependent tyrosine phosphorylation of the viral membrane protein A36R. We found that a localized outside-in signaling cascade induced by the viral membrane protein B5R is required to potently activate Src and induce A36R phosphorylation at the plasma membrane. In addition, Src-mediated phosphorylation of A36R regulated the ability of virus particles to recruit and release conventional kinesin. Thus, Src activity regulates the transition between cytoplasmic microtubule transport and actin-based motility at the plasma membrane.

Vasodilator‐stimulated phosphoprotein (VASP) is involved in multiple actin‐mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F‐actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F‐actin assembly and with SRF activation by wild‐type VASP. These VASP mutants also inhibit actin‐based motility of Vaccinia virus and Shigella flexneri . VASP‐induced F‐actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia‐mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo . These findings show that VASP and mDia function cooperatively downstream of Rho to control F‐actin assembly and SRF activity.

Paramount to the success of persistent viral infection is the ability of viruses to navigate hostile environments en route to future targets. In response to such obstacles, many viruses have developed the ability of establishing actin rich-membrane bridges to aid in future infections. Herein through dynamic imaging of HIV infected dendritic cells, we have observed how viral high-jacking of the actin/membrane network facilitates one of the most efficient forms of HIV spread. Within infected DC, viral egress is coupled to viral filopodia formation, with more than 90% of filopodia bearing immature HIV on their tips at extensions of 10 to 20 µm. Live imaging showed HIV filopodia routinely pivoting at their base, and projecting HIV virions at µm.sec⁻¹ along repetitive arc trajectories. HIV filopodial dynamics lead to up to 800 DC to CD4 T cell contacts per hour, with selection of T cells culminating in multiple filopodia tethering and converging to envelope the CD4 T-cell membrane with budding HIV particles. Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization. Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance. Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks. Dendritic cells represent a unique cell type with respect to HIV, as they are the first point of contact for the virus in the genital mucosa and have the ability to spread virus efficiently in very low numbers to the primary HIV target, CD4 T cells. During the primary immune response, dendritic cells work in small numbers to make numerous and repetitive contacts, in order to filter and communicate with appropriate CD4 T cells. Thus HIV is hypothesized to be hijacking the same DC-CD4 T cell communication. Attempts to observe how HIV would achieve this have largely been limited, as introduction of imaging markers in the virus has often led to significant viral attenuation. Herein by using novel HIV constructs that permit imaging of HIV in infected dendritic cells, we observed newly forming HIV virions on the tips of long finger-like projections known as filopodia. In real-time imaging filopodia pivoted at their base and moved virions along trajectories that led to numerous CD4 T cell contacts. By manipulating filopodial formation we conclude the location of the virus on long filopodial tips allows the virus to corrupt the promiscuous dendritic cell to CD4 T cell contacts for efficient viral spread.