Search

Infection of mammalian cells with Semliki Forest virus requires the endocytosis of the virus, its delivery to prelysosomal endosomes, and fusion of the viral envelope with the endosome membrane. Previous studies have indicated that the low endosomal pH triggers a conformational change in the viral spike glycoproteins rendering them fusogenic. In this paper, we demonstrate an additional factor(s) which regulates virus fusion in endosomes. We found that Semliki Forest virus is unable to penetrate or infect baby hamster kidney (BHK-21) cells grown in medium containing reduced Na+ concentrations. Virus endocytosis and degradation are nearly normal, the virus is transported to endosomes where a characteristic low pH-induced loss of trypsin-sensitivity of the E1 spike glycoprotein occurs. Nevertheless, the viral envelope fails to fuse with the endosomal membrane and the viral RNA is not released into the cytosol. As judged by the uptake of the voltage-sensitive probe [3H]triphenylmethyl phosphonium we observed a close correlation between conditions which inhibit virus infection and which cause depolarization of the cells. We propose that in intact cells, the fusion of Semliki Forest virus with the endosome membrane depends not only on acidic endosomal pH, but also on the maintenance of the potential.

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region. The deduced amino acid sequence demonstrates that lgp120 contains a putative signal peptide, 18 sites for N-linked glycosylation, a single membrane-spanning segment, and a short (11 amino acid) cytosolic tail. The sequence suggests a distinct domain organization, with two luminal glycosylated regions separated by a nonglycosylated proline-rich region. Proteolysis in detergent showed that the protein was not intrinsically resistant to exogenous or endogenous proteases. The N-linked oligosaccharides on lgp120, tetraantennary structures with two lactosamine repeats on one of the branches, were not different from those of glycoproteins on the plasma membrane. lgp120 was similar in its domain organization and portions of its amino acid sequence to the avian 100-kDa lysosomal membrane protein LEP100 [Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J., Siegel, N. R. & Somerville, D. (1988) J. Cell Biol. 106, 61-67], and to a distinct 110-kDa lysosomal membrane protein (lgp110) that colocalizes with lgp120. The similarities between lysosomal membrane glycoproteins from diverse species, coupled with the fact that at least two distinct lysosomal membrane glycoproteins are expressed in a single species, indicate the existence of a conserved family of glycoproteins enriched in the lysosomal membrane.

We previously isolated cDNA clones from a human monocyte library that encoded one member of a family of low‐affinity surface receptors for the Fc domain of IgG (hFcRII‐A). To investigate possible structural and functional heterogeneity among these receptors, we have now isolated two additional cDNAs (hFcRII‐B and hFcRII‐C) from a human placental library, placenta being a good source of FcR‐bearing macrophages and epithelial cells. Three cDNAs encoded related but distinct transmembrane glycoproteins containing two immunoglobulin‐like domains; however, transfected cells produced receptors that were indistinguishable on the basis of ligand binding or reactivity with anti‐hFcRII monoclonal antibodies. The sequences of hFcRII‐A and ‐B were most closely related and were identical except for several amino acid substitutions and one small internal deletion. While the ectodomain of hFcRII‐C was identical to hFcRII‐B, its cytoplasmic tail was unrelated but highly homologous to the corresponding domain of the receptor isoform (mFcRII‐B2) found in murine macrophages. Thus, human FcRII may be derived from at least two alternatively spliced genes. Northern blots revealed little difference in the pattern of expression of hFcRII isoforms among various myeloid and lymphoid cells or cell lines. However, the blots‐‐as well as in situ hybridization and immunohistochemistry‐‐demonstrated that hFcRII‐C (along with a second monocyte marker, the c‐fms encoded CSF‐1 receptor) was expressed in placental syncytiotrophoblasts. Since syncytiotrophoblasts comprise the IgG‐transporting epithelium of the placental villus, these findings suggest that FcR found in the immune system and in certain epithelia may be structurally or functionally related.