THE ROLE OF MYOSIN VI IN BACULOVIRUS INFECTION. David E. Anstey1, Kathryn G. Miller1, Biology Department, Washington University, St. Louis, MO1.
Myosin VI is an unusual member of the Myosin family because these motors move towards the pointed (-) end of actin filaments, the direction opposite of all other classes of myosin. Myosin VI is known to be crucial in the localization of actin assembly proteins and is possibly involved in cross-linking actin filaments in Drosophila melanogaster (Dm) spermatid development (Rogat and Miller 2002; Noguchi et al 2006). Recently, Dr. Loy Volkman’s lab has shown that filamentous actin is recruited to the nucleus of Lepidopteran cells during baculovirus viral infection and is necessary for nucleocapsid morphogenesis and viral progeny production. The lab has also determined that another protein, which we believe to be Myosin VI, is also recruited to the nucleus of Lepidopteran cells. We plan to knock out Myosin VI function and determine the effect of loss if myosin VI function on infection. Our hypothesis is that Myosin VI is needed for viral progeny production.
We are currently in the process of proving that Myosin VI is the protein that is being recruited to the nucleus. Western blots using antibodies against Dm myosin VI and expression of GFP-Dm myosin VI indicate that the Lepidopteran protein we detect and Dm Myosin VI behave similarly during baculovirus infection. These data suggest that they are orthologs. We are still attempting to clone and fully sequence the Lepidopteran myosin VI ortholog and demonstrate that it is Myosin VI, based on homology in conserved regions specific to Myosin VI. We have used a variety of techniques including RT_PCR and RACE to isolate portions of this gene from RNA from infected Lepidopteran cells. So far we have cloned and sequenced 2000 base pairs, more than half of the entire gene. Our current focus is on isolating and sequencing the remainder of the gene. Once we have a complete sequence, we will be able to design a GFP fusion to follow Lepidopteran Myosin VI during infection and, through RNAi knock-down and dominant negative molecule expression, determine the effect of Myosin VI loss of function on infection.