The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis-and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-␤ family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-␣/␤ (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

The Merck pneumococcal (Pn) enzyme-linked immunosorbent assays (ELISAs) for measuring antibodies to 12 serotypes (serotypes 1, 3, 4, 6B, 7F, 8, 9V, 12F, 14, 18C, 19F, and 23F) were validated in 1999. Merck Laboratories developed the Pn assays using 10 g/ml C polysaccharide, 100 g/ml Pn polysaccharide (PnPs) 25, and 100 g/ml PnPs 72 for preadsorption of samples, standards, and controls in order to improve the specificity to the Pn serotypes in the vaccine. The Pn assays utilize postimmunization sera obtained from subjects immunized with PNEUMOVAX 23 as standards for measuring immunoglobulin G concentrations in sera obtained from vaccine clinical trials with adults and infants. This material was calibrated to the Pn reference standard serum, 89SF, subjected to the Merck Pn ELISA adsorbants. Comparisons were made between the Merck Pn assay and the international Pn assay, showing moderate agreement between the two assay formats. This work describes the test procedures and operating characteristics of the Merck Pn assays and the results of experiments performed to compare the Merck Pn ELISAs to the international Pn ELISAs.PNEUMOVAX 23 (pneumococcal [Pn] polyvalent vaccine), which was licensed in 1983, is a sterile, liquid vaccine for intramuscular or subcutaneous injection. It consists of a mixture of highly purified capsular polysaccharides from 23 of the most prevalent or invasive serotypes of Streptococcus pneumoniae, including the six serotypes (serotypes 6B, 9V, 14, 19A, 19F, and 23F) that most frequently cause invasive drug-resistant Pn infections among children and adults in the United States (1). Bacterial capsular polysaccharides (Ps) induce antibodies primarily by T-cell-independent mechanisms (15); therefore, the antibody response to most Pn capsular polysaccharide types is generally poor or inconsistent in children Ͻ2 years of age, whose immune systems are immature and unable to respond effectively to the polysaccharide antigens.The immunogenicity of PNEUMOVAX 23 in early clinical trials was determined using a radioimmunoassay measuring antibodies to the Pn polysaccharides (PnPs) (14). However, due to the practicality of supporting large-scale testing, the enzyme-linked immunosorbent assay (ELISA) became the preferred method for estimating antibody concentrations. In this respect, the first-generation ELISA showed a poor correlation of antibody concentration with the efficacy of the vaccines. This observation was attributed to an overestimation of the serotype-specific PnPs antibody concentrations due to the presence of contaminating C Ps in the antigen preparations used in the ELISA (4, 6). Subsequently, a second-generation ELISA was developed by taking steps to neutralize C Ps antibodies in the serum prior to ELISA measurements. These Pn assays, utilizing the steps with preadsorption of test sera with C Ps, were compared in a multicenter study (11). The study compared results among 12 laboratories, with each laboratory testing the same series of 48 Pn paired adult serum specimens from 24 individuals (q...

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