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Adam P Geballe

Publications and source records attributed to Adam P Geballe.

12 recordsLinked to original sources
PubMed2006

her-2 upstream open reading frame effects on the use of downstream initiation codons.

The her-2 (neu, erbB-2) oncogene encodes a 185-kDa transmembrane receptor tyrosine kinase. HER2 overexpression occurs in numerous primary human tumors and contributes to 25-30% of breast and ovarian carcinomas. Synthesis of HER2 is controlled in part by an upstream open reading frame (uORF) present in the transcript. We used synthetic capped and polyadenylated mRNAs containing sequences derived from the 5' region of the her-2 transcript fused to a firefly luciferase (LUC) reporter to examine this uORF's effect on translation in cell-free systems derived from reticulocytes, wheat germ and Neurospora crassa, and in RNA-transfected HeLa cells. The uORF reduced translation of the downstream cistron in all systems. [(35)S]Met labeling of in vitro translation products obtained indicated that the uORF also affected downstream start-site selection. Primer extension inhibition (toeprint) assays of ribosomes loaded at initiation codons in reticulocyte lysates indicated that the uORF affected the interaction of ribosomes with the primary her-2 AUG codon.

Animals↗
PubMed2006

Binding and nuclear relocalization of protein kinase R by human cytomegalovirus TRS1.

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVDeltaE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2alpha kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVDeltaE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVDeltaE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVDeltaE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVDeltaE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2alpha.

Cytomegalovirus↗
PubMed2006

Double-stranded RNA binding by a heterodimeric complex of murine cytomegalovirus m142 and m143 proteins.

In response to viral infection, cells activate a variety of antiviral responses, including several that are triggered by double-stranded (ds) RNA. Among these are the protein kinase R and oligoadenylate synthetase/RNase L pathways, both of which result in the shutoff of protein synthesis. Many viruses, including human cytomegalovirus, encode dsRNA-binding proteins that prevent the activation of these pathways and thereby enable continued protein synthesis and viral replication. We have extended these analyses to another member of the beta subfamily of herpesviruses, murine cytomegalovirus (MCMV), and now report that products of the m142 and m143 genes together bind dsRNA. Coimmunoprecipitation experiments demonstrate that these two proteins interact in infected cells, consistent with their previously reported colocalization. Jointly, but not individually, the proteins rescue replication of a vaccinia virus mutant with a deletion of the dsRNA-binding protein gene E3L (VVDeltaE3L). Like the human cytomegalovirus dsRNA-binding protein genes TRS1 and IRS1, m142 and m143 are members of the US22 gene family. We also found that two other members of the MCMV US22 family, M23 and M24, encode dsRNA-binding proteins, but they do not rescue VVDeltaE3L replication. These results reveal that MCMV, like many other viruses, encodes dsRNA-binding proteins, at least two of which can inhibit dsRNA-activated antiviral pathways. However, unlike other well-studied examples, the MCMV proteins appear to act in a heterodimeric complex.

Dimerization↗
PubMed2005

Double-stranded RNA binding by human cytomegalovirus pTRS1.

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes rescue replication of vaccinia virus (VV) that has a deletion of the double-stranded RNA binding protein gene E3L (VVDeltaE3L). Like E3L, these HCMV genes block the activation of key interferon-induced, double-stranded RNA (dsRNA)-activated antiviral pathways. We investigated the hypothesis that the products of these HCMV genes act by binding to dsRNA. pTRS1 expressed by cell-free translation or by infection of mammalian cells with HCMV or recombinant VV bound to dsRNA. Competition experiments revealed that pTRS1 preferentially bound to dsRNA compared to double-stranded DNA or single-stranded RNA. 5'- and 3'-end deletion analyses mapped the TRS1 dsRNA-binding domain to amino acids 74 through 248, a region of identity to pIRS1 that contains no homology to known dsRNA-binding proteins. Deletion of the majority of this region (Delta86-246) completely abrogated dsRNA binding. To determine the role of the dsRNA-binding domain in the rescue of VVDeltaE3L replication, wild-type or deletion mutants of TRS1 were transfected into HeLa cells, which were then infected with VVDeltaE3L. While full-length TRS1 rescued VVDeltaE3L replication, deletion mutants affecting a carboxy-terminal region of TRS1 that is not required for dsRNA binding failed to rescue VVDeltaE3L. Analyses of stable cell lines revealed that the carboxy-terminal domain is necessary to prevent the shutoff of protein synthesis and the phosphorylation of eIF2alpha after VVDeltaE3L infection. Thus, pTRS1 contains an unconventional dsRNA-binding domain at its amino terminus, but a second function involving the carboxy terminus is also required for countering host cell antiviral responses.

Animals↗
PubMed2004

The effect of eukaryotic release factor depletion on translation termination in human cell lines.

Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.

Alkaline Phosphatase↗
PubMed2004

Influenza A pneumonia presenting as progressive focal infiltrates in a stem cell transplant recipient.

BACKGROUND: Stem cell transplant recipients are susceptible to pulmonary infections, including influenza A. Typically, isolated influenza pneumonia has a diffuse, interstitial infiltrate pattern. OBJECTIVES: To describe the unusual clinical and radiographic course of influenza A pneumonia in a stem cell transplant recipient. STUDY DESIGN: Case report in which microbiologic assays, bronchoscopic and pathologic specimens are obtained. RESULTS: We describe a patient with influenza A pneumonia 8 months following a peripheral blood stem cell transplant who presented with minimal respiratory symptoms and rapidly progressing, focal pulmonary infiltrates. The large size and appearance of the masses have not been reported before in a patient with isolated influenza. CONCLUSION: This case highlights the differences of presentation and importance of early diagnosis and treatment of immunocompromised patients infected with influenza.

Adult↗
PubMed2004

Evasion of cellular antiviral responses by human cytomegalovirus TRS1 and IRS1.

During infection with human cytomegalovirus (HCMV), cellular protein synthesis continues even as viral proteins are being synthesized in abundance. Thus, HCMV may have a mechanism for counteracting host cell antiviral pathways that act by shutting off translation. Consistent with this view, HCMV infection of human fibroblasts rescues the replication of a vaccinia virus mutant lacking the double-stranded RNA-binding protein gene E3L (VVdeltaE3L). HCMV also prevents the phosphorylation of the eukaryotic translation initiation factor eIF-2alpha, the activation of RNase L, and the shutoff of viral and cellular protein synthesis that otherwise result from VVdeltaE3L infection. To identify the HCMV gene(s) responsible for these effects, we prepared a library of VVdeltaE3L recombinants containing HCMV genomic fragments. By infecting nonpermissive cells with this library and screening for VV gene expression and replication, we isolated a virus containing a 2.8-kb HCMV fragment that rescues replication of VVdeltaE3L. The fragment comprises the 3' end of the J1S open reading frame through the entire TRS1 gene. Analyses of additional VVdeltaE3L recombinants revealed that the protein encoded by TRS1, pTRS1, as well as the closely related IRS1 gene, rescues VVdeltaE3L replication and prevent the shutoff of protein synthesis, the phosphorylation of eIF-2alpha, and activation of RNase L. These results demonstrate that TRS1 and IRS1 are able to counteract critical host cell antiviral response pathways.

Animals↗
PubMed2002

Interleukin-1-mediated inhibition of cytomegalovirus replication is due to increased IFN-beta production.

Previous studies have demonstrated that the intercellular spread of cytomegalovirus (CMV) is reduced in marrow stromal cells that either secrete interleukin-1 (IL-1) or are treated with exogenous IL-1. Here, we report that IL-1-treated marrow stromal cells and fibroblasts, when infected with CMV, produce decreased amounts of infectious progeny virus. CMV-infected cells treated with IL-1 contained more interferon-beta (IFN-beta) mRNA at 24 h postinfection compared with untreated, infected cells. IFN-beta protein secreted into fibroblast culture supernatants increased from 46 +/- 1 IU/ml in untreated, infected cells to 116 +/- 5 IU/ml in IL-1-treated infected cells. When IFN-beta activity was inhibited, using blocking antibodies to either the cytokine or the IFN-alpha/beta receptor, the addition of IL-1 no longer limited viral spread. Furthermore, viral spread in nonIL-1-treated cultures was inhibited by the addition of recombinant IFN-beta. These studies suggest that IL-1 functions to limit CMV spread by increasing the expression of IFN-beta, which in turn reduces production of infectious virus.

Antiviral Agents↗
PubMed2002

Complementation of vaccinia virus lacking the double-stranded RNA-binding protein gene E3L by human cytomegalovirus.

The cellular response to viral infection often includes activation of pathways that shut off protein synthesis and thereby inhibit viral replication. In order to enable efficient replication, many viruses carry genes such as the E3L gene of vaccinia virus that counteract these host antiviral pathways. Vaccinia virus from which the E3L gene has been deleted (VVDeltaE3L) is highly sensitive to interferon and exhibits a restricted host range, replicating very inefficiently in many cell types, including human fibroblast and U373MG cells. To determine whether human cytomegalovirus (CMV) has a mechanism for preventing translational shutoff, we evaluated the ability of CMV to complement the deficiencies in replication and protein synthesis associated with VVDeltaE3L. CMV, but not UV-inactivated CMV, rescued VVDeltaE3L late gene expression and replication. Thus, complementation of the VVDeltaE3L defect appears to depend on de novo CMV gene expression and is not likely a result of CMV binding to the cell receptor or of a virion structural protein. CMV rescued VVDeltaE3L late gene expression even in the presence of ganciclovir, indicating that CMV late gene expression is not required for complementation of VVDeltaE3L. The striking decrease in overall translation after infection with VVDeltaE3L was prevented by prior infection with CMV. Finally, CMV blocked both the induction of eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and activation of RNase L by VVDeltaE3L. These results suggest that CMV has one or more immediate-early or early genes that ensure maintenance of a high protein synthetic capacity during infection by preventing activation of the PKR/eIF2alpha phosphorylation and 2-5A oligoadenylate synthetase/RNase L pathways.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA.

Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.

Amino Acid Sequence↗