The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.

Recombinant baculoviruses that express the M2 protein from the genes of either the amantadine-sensitive, influenza A/Ann Arbor/6/60 virus or a laboratoryderived, amantadine-resistant mutant of this virus were constructed. Addition of amantadine or rimantadine at 2 ~tg/ml to cultures of Sf9 cells infected with the recombinant baculoviruses increased the yield of the M2 protein from the amantadine-sensitive virus approximately 10-fold, but did not increase the yield of the M2 protein from the amantadine-resistant virus. Flow cytometry demonstrated that the increased production of M2 in the presence of amantadine resulted in increased cell surface expression of the M2 protein.Pulse-chase experiments indicated that whereas the rate of synthesis of the M2 protein increased in the presence of amantadine, the M2 protein was stable in both the presence and absence of amantadine. Addition of amantadine to Sf9 cells as late as 72 h after infection with the recombinant virus increased the production of M2 protein. These data suggest that the M2 protein exerts some biological activity in Sf9 cells.

In the presence of the methyltransferase inhibitor 3-deazaadenosine (3DA-Ado) the production of infectious Autographa californica nuclear polyhedrosis virus (AcMNPV) in tissue culture was only slightly affected, while the synthesis of very late proteins (polyhedrin and pl0) was abolished. The synthesis of the influenza virus proteins NS1 and HA, expressed under the polyhedrin promoter, was also abolished by 3DA-Ado. Furthermore, 3DA-Ado interfered with the shut-off of early and late AcMNPV proteins. Most of these results were also obtained with 5-azadeoxycytidine (5A-dCyt). In cells in which NS1 was produced abundantly, at least one specific AcMNPV protein was not synthesized. However, if the production of NS1 was inhibited by 3DA-Ado, or if HA was synthesized instead, this AcMNPV protein showed up normally.