Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (DeltaUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the DeltaUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or DeltaUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

While influenza A viral RNA is known to act as a template for the synthesis of both viral mRNA and complementary cRNA, the latter has been observed so far only to function as an intermediate in replication and give rise to progeny vRNA molecules. Here it is shown that the cRNA promoter is also capable of initiating viral mRNA synthesis, similar to vRNA-promoted transcription adhering to the cap-snatching mode of primer recruitment. Detection of cRNA promoted transcription required an inversion of the reporter gene coding sequence plus relocation of the viral polyadenylation signal. Construction of cRNA promoter variants through RNA polymerase I reverse genetics allowed us to determine the RNA polymerase-associated, base-paired conformation in a reporter gene read-out system. It again turned out to adhere to the "corkscrew" model, similar, but slightly different in its binding interactions from the corresponding vRNA conformation. The observation of two transcription reactions, initiated in either direction from influenza vRNA and cRNA template molecules, allowed us to construct bicistronic, ambisense RNA molecules for simultaneous expression of two proteins from a single segment of viral RNA.