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An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of I8galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when ,1-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or fl bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of ,l-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 108 cells.

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The currently accepted model for measles virus (MV) transcription and replication assumes the nucleocapsid (NP) protein to possess the ability to bind to RNA, to other NP molecules, and to the phosphoprotein (P) during ribonucleocapsid (RNP) assembly, as well as to the matrix protein (M) during virion assembly. We have cloned the MV NP open reading frame and have expressed the protein in bacteria as a fusion with glutathione-S-transferase (GST). Affinity purified GST-NP fusion protein has been used as a probe to examine the interaction of NP with [35S] methionine labeled proteins from MV-infected cells. We have demonstrated definite and specific interactions between NP and itself and between NP and P, but have been unable to demonstrate any interaction between NP and M. We have been able to provide independent confirmation of this pattern of interaction using the yeast two-hybrid assay. We have, in addition, been able to map the domains of NP involved in these interactions by assays using sets of amino- and carboxy-terminal deletion mutants of GST-NP. The NP-NP interaction domain was found to reside in the highly conserved middle and amino-terminal domains of the protein. The hyper-variable carboxy-terminus and the conserved middle domain appear to constitute separate and independent sites for the binding of P to NP. The significance of these findings in regard to MV transcription and replication is discussed.

An outbreak of Serratia marcescens infections occurred in a university tertiary-care hospital. Alcohol-free chlorhexidine solutions were contaminated with S marcescens. The majority of patient and chlorhexidine strains had similar pulsed field-gel electrophoresis banding patterns. Chlorhexidine was recalled, and the rate of S marcescens isolation returned to baseline. Chlorhexidine without alcohol should not be used as an antiseptic.

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