Robert F. Garry

Na+ and K+ concentrations and the regulation of protein synthesis in Sindbis virus-infected chick cells.

Abstract Selective inhibition of host-specified protein synthesis in Sindbis virus-infected chick cells occurs at the level of the initiation of translation of host cell mRNAs. This inhibition is temporally correlated with an increase in the intracellular Na + concentration and a decrease in the intracellular K + concentration. Raising or lowering the NaCl concentration of the medium in which uninfected chick cells are incubated also inhibits protein synthesis, interferes with the initiation of translation of cellular mRNAs, and changes the intracellular monovalent cation concentrations. Lowering the NaCl concentration of the medium decreases intracellular K + , while raising the extracellular NaCl concentration increases both intracellular Na + and K + . The selective inhibition of host cell protein synthesis, both by Sindbis virus and by altered NaCl media, appears to result from changes in the intracellular na + and K + concentrations. Under conditions of decreased K + and/or increased Na + , viral and a few host mRNAs (such as that for chick actin), which apparently possess higher affinity for some component(s) of the initiation complex (high affinity mRNAs), can initiate while the majority of cellular mRNAs (low affinity mRNAs) cannot.

Induction of stress proteins in Sindbis virus- and vesicular stomatitis virus-infected cells

Two proteins which are related to certain proteins induced by hyperthermia (heat shock proteins; hsp) are synthesized during lytic infection of chick embryo (CE) cells by Sindbis virus or vesicular stomatitis virus (VSV). Incubation of the infected cells at elevated temperature further increased the rate of synthesis of these proteins. The stress proteins induced by Sindbis virus had different mobilities on SDS-polyacrylamide gels compared to related stress proteins induced in mock-infected CE cells. Induction of the stress proteins in Sindbis virus- and VSV-infected CE cells was actinomycin D sensitive. Kinetic studies indicated that induction of the stress proteins is an early event during infection. The lytic virus-induced selective termination of host protein synthesis did not affect the synthesis of these proteins. Furthermore, the synthesis of these virus-induced stress proteins was resistant, relative to the synthesis of most host proteins, to alterations in the intracellular concentrations of Na+ and K+. The synthesis of a protein related to a major low-molecular-weight hsp of CE cells was not induced after Sindbis virus or VSV infection. Immunoprecipitation experiments and sedimentation analyses demonstrated that significant levels of the capsid protein (C) of Sindbis virus and nucleocapsid protein (N) of VSV are physically associated with a hsp in lysates of infected CE cells.

Transformation parameters induced in chick cells by incubation in media of altered NaCl concentration

Abstract Lowering the NaCl concentration of the medium in which normal chick cell cultures are incubated causes them to enlarge, vaculate, and appear morphologically similar to cells transformed by the Bryan strain of Rous sarcoma virus (RSV). Raising the NaCl concentration of the medium causes cells to become round or spindle shaped and to appear morphologically similar to cells transformed by the Schmidt-Ruppin strain of RSV. Uninfected chick cell cultures incubated in either low or high NaCl medium also express many characteristics of transformed cells. They lose contact inhibition of growth and movement, grow to higher saturation densities, synthesize reduced amounts of fibronectin, exhibit increased lectin agglutinability, transport increased amounts of hexoses, have reduced levels of succinic dehydrogenase activity, produce increased amounts of lactate and pyruvate, and can be serially passaged in culture significantly longer than normal chick fibroblasts. We were not able to induce normal chick cells to exhibit all transformation parameters by incubation an altered NaCl media: the cells did not grow in soft agar or in low serum medium. Cells transformed by the Bryan and Schmidt-Ruppin strains of RSV exhibit strain-specific changes in their intracellular Na+ and K+ concentrations. Cells incubated in altered NaCl media exhibit altered intracellular monovalent cation concentrations similar to those seen in the cells they resemble morphologically. Results presented in this publication identify a possible new transformation parameter, alteration of the intracellular Na+ and K+ concentrations, and suggest that many, but not all transformation parameters may be consequences of the altered intracellular monovalent cation concentrations.

Na+ and K+ concentrations and the regulation of the interferon system in chick cells

Abstract Media of altered NaCl concentrations were used to change the intracellular concentrations of Na + and K + in normal chick embryo fibroblasts (CEF) to resemble those which occur after lytic virus infection. In these modified media, CEF produced normal amounts of interferon although the bulk of cellular protein synthesis was inhibited. Since viral mRNAs can be translated under these conditions, interferon mRNA appears to resemble viral mRNAs. Vesicular stomatitis virus (VSV) replication was not inhibited when interferon-pretreated cells were incubated in altered NaCl media, indicating that the mRNA for at least one interferon-induced antiviral protein is a more typical host mRNA. When interferon-producing cells were incubated in altered NaCl media, normal termination of interferon production did not occur. This suggests that the regulatory protein which shuts off interferon production also has a typical cellular mRNA. In addition, interferon treatment did not change the intracellular concentrations of Na + or K + but did prevent the changes which occur as a consequence of lytic VSV infection.

Membrane-mediated alterations of intracellular Na+ and K+ in lytic-virus-infected and retrovirus-transformed cells.

Infection of chick-embryo fibroblasts and other cells by certain animal viruses results in alterations in the intracellular concentrations of Na+ and K+. Dramatic alterations in monovalent-cation concentrations of lytic-virus-infected cells may favor the synthesis of viral proteins over cellular proteins. More subtle alterations in retrovirus-transformed cells may result in the expression of many morphological and biochemical changes associated with the transformed phenotype.

Differential effects of ouabain on host-and sindbis virus-specified protein synthesis

Abstract Ouabain, a specific inhibitor of the membrane-associated Na + /K + -ATPase (sodium pump), has a differential effect on protein synthesis in Sindbis virus-infected and uninfected chick embryo fibroblast (CEF) cultures. At low concentrations, cell-specified protein synthesis is inhibited while Sindbis virus-directed protein synthesis is stimulated. At higher ouabain concentrations, which inhibit protein synthesis in uninfected CEF cells 95% or more, viral protein synthesis continues. Ouabain treatment of uninfected CEF cells raises the intracellular Na + concentration and lowers the intracellular K + concentration. The effect of Sindbis virus infection is similar. The changes in intracellular Na + and K + concentrations appear responsible for the selective inhibition of the translation of host cell mRNAs in Sindbis virus-infected or ouabain-treated CEF.

Effect of incubation time on the generation of defective-interfering particles during undiluted serial passage of sindbis virus in Aedes albopictus and chick cells

Abstract Increasing the length of the incubation period enhances the generation of Sindbis virus (SV) defective-interfering (DI) particles and other genetic variants in chick embryo fibroblast (CEF) cells. SV DI particles were also generated by serial undiluted passage in Aedes albopictus (mosquito) cells at 48-hr intervals. The cyclic variation in SV titer characteristic of DI particles began in passage 5. The presence of DI particles in certain virus stocks was confirmed by demonstrating that virus from these passages interfered with the replication of plaque-purified virus and caused the appearance of abnormally small intracellular, SV-specified RNAs. SV DIs generated in A. albopictus cells did not interfere with the replication of SV in chick cells, nor did SV DIs generated in chick cells interfere in A. albopictus cells. In addition to DI particles, certain of the serially passaged Sindbis virus stocks contained temperature-sensitive (ts) and small and medium plaque variants.

Chicken fetal antigens: Role as cell surface receptors for sindbis virus hemagglutinin

Abstract Sindbis virus attaches to and agglutinates red blood cells from fetal and developing chickens that express chicken fetal antigens (CFA). Erythrocytes from adult chickens that do not express CFA do not contain the receptor site for the Sindbis virus hemagglutinin. The attachment of Sindbis virus to erythrocytes of different avian species indicates that Sindbis virus attachment occurs only when a specific CFA determinant, CFA determinant 9, is expressed. Erythrocytes reacted with antiserum directed against CFA determinant 9 fail to attach to or to be agglutinated by Sindbis virus. These results explain the failure of Sindbis virus to agglutinate adult chicken erythrocytes and identify the receptor for the Sindbis virus hemagglutinin.

Secretion of a virus-regulated factor by clonal variants of reticuloendotheliosis virus-transformed hematopoietic cells

Abstract Reticuloendotheliosis virus (REV-T) is an avian acute leukemia virus which transforms hematopoietic cells which express bursal cell determinants in vivo and in vitro . REV-T-transformed hematopoietic cell lines spontaneously give rise to stable variants that secrete a factor which results in the formation of a halo surrounding colonies formed in soft agar. Expression of this phenotype does not correlate with helper virus production, growth rate or morphology, secretion of immunoglobulin, or the tumorigenic potential of the cells. The secretion of this factor appears to be regulated by the genetic information of REV-T.