Kathryn C. Stallcup

Cytochalasin B inhibits the maturation of measles virus

The release of measles virus was studied in the presence of cytochalasin B (CB), a drug that disrupts actin microfilaments. In the presence of CB, infected cells accumulated infectious virus while virus released from these cultures decreased drastically (up to 99% inhibition). Electron micrographs showed that viral buds were reduced and had an unusual distribution along the cell membrane in CB-treated cultures. CB inhibition of released virus occurred rapidly (within 30 min) and to a full extent even when the drug was added during the final 2 hr of a 48-hr replicative cycle. CB inhibition of cellular functions is reversible and, similarly, inhibition of virus release could be almost completely reversed within 30 min after the drug was removed. Since CB can also inhibit sugar transport and protein glycosylation, 2-deoxy-D-glucose (DG) was used to study the manifestations of glycosylation inhibition. DG inhibited virus production only when added during the first one-third of the replicative cycle and inhibited cell-associated and released virus to an equal extent. Cytochalasin D, which disrupts microfilaments without affecting protein glycosylation, caused an inhibition of virus release analogous to the inhibition caused by CB. Thus, alteration of microfilament structure alters the normal budding process of measles virions. This suggests that microfilaments may play a role in the release of budding virions.

[8] Purification of murine MHC antigens by monoclonal antibody affinity chromatography

Publisher Summary This chapter discusses the purification of murine major histocompatibility complex (MHC) antigens by monoclonal antibody affinity chromatography. The growing understanding of the role of MHC antigens in transplantation and immune system recognition has resulted in considerable interest in the functional and structural characteristics of these cell-surface glycoproteins. Monoclonal antibody affinity chromatography has proved to be a rapid and efficient means of purifying relatively large amounts of both class I and class II murine MHC antigens. There is a successfully use of four monoclonal antibodies as affinity reagents. The same general approach is applicable to the purification of a variety of MHC antigens using different monoclonal antibodies. The antigens purified in this way retain serological and biological activity and prove to be useful for studying the structure and function of these molecules.

The replication of measles virus in the presence of tunicamycin

Abstract The glycoproteins of measles are involved in a number of biological activities, including infectivity and cell fusion. In order to study the role of carbohydrates in the function of viral glycoproteins, tunicamycin (TM) was used to inhibit the addition of sugars to glycoproteins. One microgram per milliliter of the drug, administered 2 hr postinfection to cell cultures infected with virus, caused a 10 3 –10 4 reduction in the production of infectious progeny virus. This inhibition occurred at either 33 or 37° and affected both the Edmonston and Joy wild-type measles strains. The release of material with the density of virions was inhibited 85–90%. Synthesis of the nonglycosylated viral proteins (L, P, NP, M) appeared to be normal in the presence of the drug, while changes were observed in the synthesis of H, the large viral glycoprotein, and F 1 a component of the fusion protein. Particles released from TM-treated cultures lacked the normal H and F 1 proteins. Virion preparations contained a polypeptide, termed p90, which migrated more slowly than H on SDS-PAGE and was also found in preparations of particles released from cultures infected in the presence of 2-deoxy- d -glucose. Normal cultures pulsed for 2 hr with [ 35 S]methionine late in infection released virions that had incorporated the label into the L, p90, P, NP, and M polypeptides.

Inhibition of normal lymphocyte responses by cell membranes.

Cell membranes bearing the appropriate antigen are known to stimulate a variety of cell-mediated immune responses. This report confirms that tumor cell membranes at doses of 2-5 micrograms protein/ml will stimulate in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). However, higher doses (50-100 micrograms protein/ml) of the same membranes completely abrogate the generation of lytic activity. Responding lymphocytes are inhibited by membranes from either syngeneic or allogeneic cells. The inhibition appears to act at a proliferative or differentiation step in the generation of the CTL response, since membranes are known to have little direct effect on the lytic phase of CTL activity. Similar doses of membranes also inhibit LPS-induced B-cell proliferation. B-Cell proliferation is inhibited equally well by allogeneic and syngeneic membranes, and membranes from normal spleen cells are as inhibitory as tumor cell membranes. The inhibitory activity copurifies with the plasma membrane. The results raise important considerations regarding the use of subcellular forms of antigen in studies of lymphocyte recognition. In addition, these data suggest that cell-cell contacts might provide signals regulating the proliferation of lymphocytes.