INDUCING SINGLE-CELL SUSPENSION OF BTI-TN5B1-4 INSECT CELLS .2. THE EFFECT OF SULFATED POLYANIONS ON BACULOVIRUS INFECTION

Sulfated polyanions can be used to rapidly induce and maintain single- cell suspensions of BTI-TN5B1-4 insect cells, a cell line which clumps in suspension. Elimination of cell clumping results in a significant increase involumetric yield of the baculovirus expression vector syste m. Sulfated polyanions, however, inhibited baculovirus infection of BT I-TN5B1-4. Data from binding studies and fusion assays suggest that th e inhibition of infection was not due to the observed reduction in vir al attachment rate but to inhibition of viral membrane fusion in the e ndosome. The three most effective polyanions for inducing single cells are dextran sulfate, pentosan sulfate, and polyvinyl sulfate. At conc entrations required for single-cell formation, dextran sulfate and pen tosan sulfate did not affect viral infection at multiplicities of infe ction greater than one plaque forming unit per cell. In contrast, poly vinyl sulfate blocked viral infection even at a high multiplicity of i nfection of 20 plaque-forming units per cell. To bypass this inhibitio n, polyvinyl sulfate can be removed by resuspending the cells in fresh medium before virus addition, and then added back to the cell suspens ion after a substantial amount of virus has been internalized. Alterna tively, polyvinyl sulfate can be neutralized with a polycation before virus addition, and an equivalent amount of polyvinyl sulfate added ba ck after most of the virus has been internalized. We present a simple mathematical model of the attachment and entry of baculovirus in BTI-T N5B1-4, which can be used to design appropriate infection regimens. (C ) 1997 John Wiley & Sons, Inc.