Replication of H1N1 influenza viruses in cultured mouse embryo brain cells: Virus strain and cell differentiation affect synthesis of proteins encoded in RNA segments 7 and 8 and efficiency of mRNA splicing

The aims of these studies are (1) to determine whether, and by what mechanism(s), underexpression of M1 and/or NS1 protein restricts replication and cytopathogenicity in mouse brain cells of human influenza viruses which are closely related to the neurovirulent WSN variant but not selected for the neurovirulent phenotype; (2) to learn, ultimately, whether similarly restricted replication in natural infections might be enough to cause direct or indirect, immunologically mediated, neuropathology. On the basis of immunostaining, we have suggested that, in “aged” mouse embryo brain (MEB) cell cultures infected with A/PR/8/34 (PR8) or A/WS/33 (WS), M1 protein expression is restricted mainly in mature astrocytes (the dominant cell type in such cultures), but not in mature oligodendrocytes or neurons. Here we show that amounts of radiolabeled M1 protein in lysates of MEB cultures infected with PR8, WS, or WSN differ in proportion to previously reported single-cycle yields of trypsin-activated infectious virions. Low or undetectable cell-ssociated M1 does not reflect accelerated degradation, but tends to be accompanied by increased M2 protein (a product of spliced mRNA7). Radiolabeled NS1 is reduced, NS2 relatively increased, in “ed” MEB cultures infected at low m.o.i. with PR8, at high m.o.i. with WS as well, but not with WSN. In contrast, actively dividing and differentiating astrocyte-enriched or “young” MEB cultures tend to produce greatly increased amounts of NS2 even though NS1 may be at “normal” levels, both relative to those in similarly infected CEF cultures. We show, in extension of comparative studies by others on permissive and abortive FPV-infected cell systems, that virus strain-, cell type-, and perhaps differentiation-dependent variations in efficiency of mRNA 7 and 8 transcription and/or splicing are primary factors controlling variable expression of M and NS proteins in mouse brain cell cultures.