Emin T. Ulug

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.

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.

Overexpression of murine phosphatidylinositol 4‐phosphate 5‐kinase type Iβ disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4‐phosphate 5‐kinases (PI4P5K) phosphorylate phosphatidylinositol 4‐phosphate [PI(4)P] to produce phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Iβ (mPIP5K‐Iβ) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand‐dependent downregulation of the colony‐stimulating factor‐1 receptor. The present study shows that enforced expression of mPIP5K‐Iβ in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2‐binding pleckstrin homology (PH) domain of phospholipase‐Cδ (PLC‐δ). Analysis of the conserved domains of mPIP5K‐Iβ led to the identification of dimerization domains in the N‐ and C‐terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K‐Iβ and the PLC‐δ‐PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K‐Iβ acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion. J. Cell. Biochem. 85: 131–145, 2002.

The role of monovalent cation transport in Sindbis virus maturation and release

Alterations in intracellular monovalent cation concentrations in Sindbis virus-infected avian cells result, in part, from a reduction in Na+/K+ ATPase (Na+ pump) activity. Inhibition of Na+ pump activity was shown previously to temporally correlate with the appearance of viral envelope proteins on the cell surface and the release of virus particles. Cells infected with envelope-defective temperature-sensitive mutants exhibited reduced Na+ pump activity at the nonpermissive temperature, where viral particles are not released. By contrast, Na+ pump activity was not inhibited in Sindbis virus-infected cells treated with tunicamycin or with antiviral serum, which block virus maturation and release. Diuretic-sensitive transport of 86Rb+, aK+ tracer, was stimulated in cells which express virus envelope proteins, but fail to release virus particles. In these cells, the furosemide-sensitive 86Rb+ influx exhibited an increase in Vmax and was responsive to changes in the extracellular concentration of NaCl. Furosemide inhibited the rapid release of virus from low salt-inhibited cells after shift to isotonic conditions. Alterations in ion transport during alphavirus infection may, therefore, facilitate the efficient release of progeny virus particles.

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.

Concise syntheses of l-α-phosphatidyl-d-myo-inositol 3-phosphate (3-PIP), 5-phosphate (5-PIP), and 3,5-bisphosphate (3,5-PIP2)

Abstract Highly efficient, asymmetric total syntheses of the title phospholipids as well as short chain and cross-linkable aminoether analogs were achieved in five to seven steps from a readily available myo -inositol derivative.

Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts

In Sindbis virus-infected avian cells the development of the cytopathic effect is correlated with the disruption of plasma membrane function. Sindbis virus inhibits the activity of the Na+K+ATPase, a membrane-associated enzyme complex which regulates intracellular monovalent cation levels. Tunicamycin, which blocks envelope protein glycosylation, prevents inhibition of Na+K+ATPase activity and the development of morphological changes in Sindbis virus-infected cells. Although inhibition of Na+K+ATPase activity is not essential for the termination of host protein synthesis, membrane-mediated events may favor the selective translation of viral proteins. The termination of host protein synthesis does not contribute to the development of these cytopathic changes in the time frame examined. In tunicamycin-treated, Sindbis virus-infected cells, unglycosylated E1 is inserted into the plasma membrane but virus release is prevented. In productively infected cells, therefore, the inhibition of Na+K+ATPase activity and the development of the cytopathic effect may result from terminal events in virus assembly and/or virus release.

Differential Regulation of the Inhibitor of Apoptosis ch-IAP1 by v-rel and the Proto-Oncogene c-rel

ABSTRACT The v-rel oncogene encoded by reticuloendotheliosis virus is the acutely transforming member of the Rel/NF-κB family of transcription factors. v-Rel is a truncated and mutated form of c-Rel and transforms cells by inducing the aberrant expression of genes regulated by Rel/NF-κB proteins. The expression of ch-IAP1, a member of the inhibitor-of-apoptosis family, is highly elevated in cells expressing v-Rel and contributes to the immortalization of cells transformed by this oncoprotein. In this study we demonstrate that the elevated expression of ch-IAP1 in v-Rel-expressing cells is due to an increased rate of transcription. The ch-IAP1 promoter was isolated, and four Rel/NF-κB binding sites were identified upstream of the transcription start site. Two κB sites proximal to the transcription start site were required for v-Rel to activate the ch-IAP1 promoter. While c-Rel also utilized these sites, a third more-distal κB site was required for its full activation of the ch-IAP1 promoter. Differences in the transactivation domains of v-Rel and c-Rel are responsible for their different abilities to utilize these sites and account for their differential activation of the ch-IAP1 promoter. Although c-Rel was a more potent activator of the ch-IAP1 promoter than v-Rel in transient reporter assays, cells stably overexpressing c-Rel failed to maintain high levels of ch-IAP1 expression. The reduction of ch-IAP1 expression in these cells correlated with the efficient regulation of c-Rel by IκBα. The ability of v-Rel to escape IκBα regulation allows for the gradual and sustained elevation of ch-IAP1 expression directly contributing to the transforming properties of v-Rel.

Alterations in focal adhesion kinase activity and associated proteins during malignant conversion of mouse keratinocytes

Focal adhesion kinase (pp125FAK) has well‐established functions in the attachment and growth of cells in culture and has been implicated as a marker of malignant progression in human tumors. To evaluate its role in the metastatic conversion of mouse skin tumors, pp125FAK activity and protein expression were examined in normal and transformed keratinocyte cell lines. Malignant mouse keratinocyte lines exhibited a reproducible increase in the specific activity of pp125FAK compared with that of nontransformed control cells. An increase in pp125FAK activity was not observed in papilloma‐derived keratinocytes, indicating that this response correlated with malignant progression of cells and not cell transformation per se. Immune complex kinase assays and metabolic labeling with [32P]orthophosphate also revealed the specific loss of pp125FAK‐associated proteins in the metastatic keratinocytes. Furthermore, immunocytochemical examination revealed an altered distribution of pp125FAK in the cells with malignant potential compared with normal and papilloma‐inducing keratinocytes. The cells with malignant potential also exhibited reduced levels of paxillin and integrin β1 as well as altered distribution of paxillin, reinforcing the notion that specific changes in the composition of focal adhesions contribute to the malignant conversion of mouse keratinocytes. Mol. Carcinog. 25:73–83, 1999.

Murine polyomavirus microRNAs promote viruria during the acute phase of infection

Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. Studies presented here use murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis reveals that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection, indicating that during infection of a natural host, PyV miRNAs function to reduce viral replication during the persistent phase of infection. Additionally, MuPyV miRNAs promote viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/-mice. These findings implicate miRNA activity in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response. Importance MicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of possible clinical relevance. Here we show a novel activity for a viral miRNA in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA in long-term persistence, that it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an effective adaptive immune response, our work also connects the in vivo activity of a PyV miRNA to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings may help to better understand how the balance between PyV and the immune response becomes altered in pathogenic states.