MEASLES-VIRUS SUBSTANCE-P RECEPTOR INTERACTIONS - JURKAT LYMPHOCYTES TRANSFECTED WITH SUBSTANCE-P RECEPTOR CDNA ENHANCE MEASLES-VIRUS FUSION AND REPLICATION

被引:24
作者
HARROWE, G
SUDDUTHKLINGER, J
PAYAN, DG
机构
[1] UNIV CALIF SAN FRANCISCO,DEPT MED,3RD & PARNASSUS AVE,BOX 0724,SAN FRANCISCO,CA 94143
[2] UNIV CALIF SAN FRANCISCO,DEPT MICROBIOL & IMMUNOL,SAN FRANCISCO,CA 94143
[3] UNIV CALIF SAN FRANCISCO,HOWARD HUGHES MED INST,SAN FRANCISCO,CA 94143
关键词
SUBSTANCE-P; SUBSTANCE-P RECEPTOR; MEASLES VIRUS; NEUROIMMUNOLOGY; VIRAL FUSION; VIRAL REPLICATIONS;
D O I
10.1007/BF00711541
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
1. We have demonstrated previously (Harrowe et al., 1990), using a lymphoblastoid cell line that constitutively expresses the substance P receptor (SPR) (Payan et al., 1984, 1986), that this receptor may facilitate measles virus (MV) fusion with these cells. In order to test this hypothesis further, a stable cell line transfected with SPR cDNA has been established, and various stages of MV infection in SPR positive and negative cells compared. 2. Jurkat cells, a human T-lymphoblastoid cell line, were transfected with a cDNA clone encoding the SPR. Cells transfected with only the plasmid were used as controls. Jurkat cells and Jurkat vector control cells (J-vo) failed to demonstrate any detectable I-125-SP binding, whereas a clonally selected population of cells transfected with SPR cDNA (J-SPR) expressed about 50,000 receptors/cell (Sudduth-Klinger et al., 1992). 3. Using the J-vo- and J-SPR-transfected cell lines, the following experiments were conducted to investigate the effect of SPR expression on MV infection. To determine if MV would preferentially attach to J-SPR as compared to J-vo, we absorbed virus to cells at 37-degrees-C for various times and measured bound MV using a fluorescence activated cell sorter (FACS). Using this approach, we found that MV bound to a greater degree to J-SPR compared with J-vo. In addition to equilibrium being reached faster for J-SPR, the total amount of bound MV was higher on J-SPR. The effect was greater at lower MOIs, suggesting that there existed multiple binding sites for MV on these cells and that the affinity is higher for those cells expressing the SPR. 4. Since binding does not necessitate a successful viral infection, we needed to know if this difference in binding reflected a difference in infection. This was demonstrated by showing an approximate twofold increase in infected cells after a 2-hr binding period with J-SPR as compared to J-vo at an MOI of 1 in an infectious cell-center assay. Moreover, when both cells types were subjected to continuous infection in culture, J-SPR-infected cells produced a seven- to ninefold increase in measles viral titer in 24 hr as compared with J-vo. The observed increase in viral titer may have resulted in more of the J-SPR cells binding virus, as indicated by our binding and infectious cell-center data, or alternatively, the virus might have entered the J-SPR cells faster and begun replication before the J-vo-infected cells. 5. The possibility of a difference in the rate of fusion was investigated using the octadecyl rhodamine B (R18) dequenching assay. This assay utilizes the ability of R18 to quench its own fluorescence at high concentrations in the viral membrane (Blumenthal et al., 1987). Upon fusion of R18-loaded virus with cell membrane, a release of quenching occurs as the dye dilutes itself in the cell's plasma membrane, causing an increase in fluorescence. This allows for a kinetic measurement of viral fusion. Employing this technique, we looked at the fusion kinetics of R18 MV absorbed to J-SPR and J-vo. After a 2-h preincubation with cells and R18-loaded virus at 4-degrees-C, the virally bound cells were warmed to 37-degrees-C in a fluorimeter and fluorescence was monitored. The results demonstrated a rapid increase in fusion with J-SPR relative to J-vo. In addition, the level of fluorescence was higher for J-SPR compared with J-vo. These data suggest that fusion was enhanced by the SPR, and per unit time, more virus was bound and fusing. 6. SPR expression in Jurkat cells facilitates and enhances MV infection. The molecular mechanisms by which this facilitation takes place are an area of ongoing investigation in this laboratory.
引用
收藏
页码:397 / 409
页数:13
相关论文
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