Kinesin-dependent movement on microtubules precedes actin-based motility vaccinia virus

Viruses are biological particles which are causative agents of various diseases and examples of which are the herpes simplex virus, adenovirus, and vaccinia virus. Infections are caused by viruses only under specific conditions wherein they are situated inside a living host cell, an event which accounts for its intracellular nature of existence and infection. In addition to this, viruses tend to maximize the usage of the cell components of the host organism in order for them to attain a high copy number inside the hosts body system. To accomplish such kind of activity, viruses need to multiply by replication and move the replicated viral particles into the different sites of the hosts body and this activity requires the utilization of the intracellular energy and components of the host such as the microtubules and dynein-dynactin complex. The aforementioned viruses, herpes simplex, adenovirus, and vaccinia virus, all utilize the microtubules and dynein-dynactin complex of the host to initiate replication and continue with the infection cycle. Furthermore, cell-to-cell spread of viruses from infected cells to non-infected cells is facilitated by the usage of diffusion and cytoskeleton movements within the host cell where the latter accounts for the transfer of relatively small viruses and the latter explains for the relocation of the viral particles through the help of actin-based motility as initiated by the hosts cytoskeleton. In order to further explore the topic of actin-based motility of viral particles, the goal of this paper is to focus on the discussion of the mechanism by which vaccinia viruses are able to elicit cell-to-cell spread. The discussion is aimed to center its attention on the exploration of the tyrosine phosphorylation of the intracellular mature virus (IMV) as explained by the activity on A36R membrane protein, WASP-interacting protein (WIP) and N-WASP, and event which results to the production of long, virus-tipped projections at the plasma membrane of the host cell which facilitates cell-to-cell movement of vaccinia viruses. The significance of this study is evident on the fact that the vaccinia virus constitutes the best-characterized model of cytoskeleton-driven viral motility and a demonstration of the additional features of these microscopic activities is important in the enhancement of present knowledge on the nature and dynamics of viral particle behavior and activity so as to prevent and control the proliferation of detrimental to lethal viral disease like the smallpox, a disease that is caused by the family of vaccinia virus.  

The experimental process employed to accomplish the goal of this research paper is composed of seven steps and these are the generation of stable GFP--actin cell lines, pEL vector constructs, preparation of the antibodies, infection, pEL-driven expression and immunoflorescence on fixed preparations, live cell imaging, analysis of the images produced, and, the construction of recombinant AR36-YdF vaccinia virus. The first step, explains the amplification via polymerase chain reaction methods of the chicken cell lines and human sarcoma cells that served as the host cells of the viruses while the second step characterized the amplification of the F13L and A36R open reading frames of vaccinia virus which were used as the vector of infection. The third step, preparation of antibodies, was accomplished by using rat monoclonal antibodies, an event which is essential in the visualization of the actin, through the usage of the anti--actin antibody AC-74, and microtubules, through the usage of pactin-mb5tubulin-EGFP. The fourth step, on the other hand, is comprised of the vaccinia infection on the host cell lines and subsequent performance of immunofloresence procedures to view the infected cells and viral particles. Live cell imaging is accomplished by utilizing a specialized camera that was able to detect the volume imaging and cell plane viewing and this step was followed by deconvolution and image reconstruction to better scrutinize the features of the images and manifest virus movements via microtubule assistance. The last step, construction of recombinant A36R-YdF vaccinia virus, was accomplished by harvesting the freeze-thawed recombinant vaccinia particles and confirming the fidelity of the recombinant through PCR methods, a step which marked the verification of the observed experimentally observed importance of actin-based viral movement for vaccinia particles.

Results show that actin tails have originated from the plasma membrane through the help of the cell-associated envelope virus (CEV) and this is in contrary to the previous observation that intracellular enveloped particles (IEVs) have nucleated actin tails. This observation is supported by the fact that electron microscopy was able to produce images that explains the distinction of IEVs from CEV at the tip of actin projections, and event which caused the confusion of the nature of actin sources. Despite the fact that researchers cannot rule out the possibility that the formation of actin filaments helping vaccinia virus during cell-to-cell spread is participated by IEVs, the notion that the mechanism by which actin-based movement of vaccinia virus is more obviously allotted to CEV is an important finding in this paper. This paper has disproven the previous assumption that actin polymerization is initiated at the periphery of plasma membrane and often with associated CEVs at the tip of the projections because it was confirmed by imaging that actin filaments only form close to the periphery of the plasma membrane. It was also clarified in this paper that actin polymerization is induced on the inner cytoplasmic surface of the plasma membrane through the help of the A36R, an integral membrane protein. Moreover, imaging analysis revealed that high quantity of virus release is achieved by vaccinia particles while maintaining low CEV retention at the plasma membrane. In general, this paper confirmed the idea that vaccinia virus makes use of microtubules and cytoskeleton to enhance its infection process.


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