Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins

It has previously been demonstrated in this laboratory that an influenza vi rus-like chloramphenicol acetyltransferase (CAT) RNA could be expressed in COS-1 cells that synthesized all ten influenza A virus-encoded proteins fro m recombinant plasmids. It was also shown that supernatant fluids harvested from these cultures contained virus-like particles (VLPs) that could deliv er an enclosed CAT RNA to MDCK cells. Here, it is shown that the levels of expression of the reporter gene in the COS-1 and/or MDCK cells can be alter ed drastically by modifying the concentrations of the recombinant plasmids transfected in the COS-1 cells. Thus, it was observed that overexpression o f NS2 reduced CAT expression in COS-1 cells, whereas overexpression of M2 a nd NS1 proteins dramatically decreased transmission of the CAT RNA to the M DCK cultures. These results are discussed with reference to the roles of th ese proteins during virus replication, From these experiments, a ratio of t ransfected plasmids was found that increased the efficiency of the previous ly described system by 50-100-fold. Under these optimized conditions, it wa s demonstrated that VLPs can be formed in the absence of neuraminidase expr ession and that these VLPs remained aggregated to each other and to cell me mbranes, Moreover, it is shown that CAT RNA transmission was dependent on s pecific interactions of the ribonucleoprotein complex with other viral stru ctural polypeptides. These data demonstrate the usefulness of this encapsid ation-packaging system for the study of different aspects of the influenza virus life-cycle.