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The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.

The 11.6-K protein of human adenovirus 2 (Ad2), which was recently renamed as adenovirus death protein (ADP), is a type III membrane glycoprotein that ultimately localizes to the nuclear membrane. ADP is encoded in the E3 transcription unit of Ad2 and migrates as a set of multiple bands in SDS-PAGE with three major forms. The corresponding gene product of adenovirus 5 (Ad5) has a slightly lower molecular weight and shows the same pattern in SDS-PAGE. We report here the covalent attachment of fatty acids to cysteine residues of ADP. In the case of Ad5-ADP all three major forms of this protein can be labeled by [3H]palmitic acid, but not by [3H]myristic acid, whereas only two [3H]palmitic acid-labeled Ad2-ADP species could be detected. The label is sensitive to treatment with 1 M hydroxylamine at pH 7 and with 20% beta-mercaptoethanol indicating that the fatty acids are linked via a thioester bond. By thin layer chromatography, the vast majority of the incorporated label was identified as palmitic acid. Two cysteine residues at the boundary between transmembrane domain and cytoplasmic tail which could serve as acceptor sites were mutated to alanine residues by site-directed mutagenesis of the cloned Ad5-ADP gene. Expression of wild-type Ad5-ADP and the resulting mutants was performed in HeLa cells using the vaccinia virus T7 expression system. As demonstrated by labeling with [3H]palmitic acid, only the mutants with one remaining cysteine residue in the cytoplasmic tail were able to incorporate [3H]palmitic acid, indicating that either could serve as acceptor site. In contrast the double cysteine mutant could not be labeled by [3H]palmitic acid, clearly demonstrating that cysteines 53 and 54 are required for palmitoylation and probably represent the palmitoylation sites in Ad5-ADP.

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants tsl and ts227, which have been analyzed previously (W.