30K Protein Research Articles

Identification of a cell surface 30 kDa protein as a candidate receptor for Hantaan virus.

Cellular receptors play an important role in virus pathogenesis. As a first step in virus infection, viruses attach to specific receptors on the surface of cells; Hantaan virus infects susceptible cells by attaching to a receptor located on the cell surface. To date, the identity of the Hantaan virus host cell receptor remains unknown. To determine the protein on the cell surface to which Hantaan virus binds, a virus overlay protein-binding assay was performed with radiolabelled virus. A 30 kDa (30K) protein was identified as a putative receptor for Hantaan virus. The specificity of virus interactions with this protein was demonstrated with a competition assay using unlabelled Hantaan virus and poliovirus. Unlabelled Hantaan virus inhibited the binding of radiolabelled Hantaan virus to this 30 kDa protein, whereas poliovirus did not. A polyclonal antibody against the 30K protein blocked binding of Hantaan virus to Vero-E6 cells and, consequently, virus infection. Blocking with the anti-30K antibody reduced virus infection of cells by 70%. These data strongly suggest that the 30 kDa surface protein is a putative receptor for Hantaan virus.

Viral genome-linked protein (VPg) controls accumulation and phloem-loading of a potyvirus in inoculated potato leaves.

The viral protein covalently linked to the 5' end of the plus-sense, single-stranded RNA genome of potyviruses (genus Potyvirus) can be an avirulence determinant in incompatible potyvirus-host combinations in which the resistance prevents systemic virus infection. The mechanism is not well known. This study shows that virus strain-specific resistance to systemic infection with Potato virus A (PVA) in Solanum commersonii is overcome by a single amino acid (aa) substitution, His118Tyr, in the viral genome-linked protein (VPg). Virus localization and other experiments revealed that Tyr118, controls phloem loading of PVA. The critical boundary may be constituted in phloem parenchyma, companion cells, or both. Tyr118 also controls the cellular level of virus accumulation in infected leaves, including phloem cells. Amino acid substitutions at three additional positions of the central part (aa 116) and C terminus (aa 185) of the VPg and of the N terminus of the 6K2 protein (aa 5) affect virus accumulation and rate of systemic infection but are not sufficient for phloem loading of PVA. These data, together with previous studies, indicate that the PVA VPg aa residues crucial for systemic infection are host specific. Also, our data and previous studies on other potyvirus-host species combinations indicate that the central part of the VPg is a domain with universal importance to virus-host interactions required for systemic invasion of plants with potyviruses.

Replication protein RepN encoded by the RC plasmid of thermophilic bacterium Thermoanaerobacterium saccharolyticum: mutational analysis and deletion mapping of domains responsible for its lethal effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif, which showed no appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for the stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.

The 42K Protein of Shallot Virus X Participates in Formation of Virus Particles

The 42K Protein of Shallot Virus X Participates in Formation of Virus Particles

Tumor Necrosis Factor-α Induces Bax-Bak Interaction and Apoptosis, Which Is Inhibited by Adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.

Plant Golgi-associated vesicles contain a novel α-actinin-like protein

By using Western blotting, immunofluorescence and immunogold labeling, a novel alpha-actinin-like protein was found in pollen and pollen tubes of Lilium davidii, a model system for cytoskeleton and Golgi apparatus study of plant. As measured by Western blotting, the molecular mass of the a-actinin-like protein was about 80 kDa. Under confocal laser scanning microscopy after immunofluorescence labeling, the distribution of the alpha-actinin-like protein appeared punctated in the cytoplasm of the pollen and pollen tubes. When double labeled, the protein was co-localized with Golgi 58K protein. In addition, some fraction of the alpha-actinin-like protein was found to co-distribute with F-actin bundles in the pollen tubes. Additional studies with immuno-gold labeling and transmission electron microscopy revealed that the alpha-actinin-like protein bound mainly to the membranes of Golgi-associated vesicles. When the pollen tubes were treated with Brefeldin A (BFA), the a-actinin-like proteins were dispersed into the cytoplasm, and the growth of pollen tubes was inhibited. After BFA was removed, the protein was reversibly recovered on the Golgi apparatus. These results suggest that the novel alpha-actinin-like protein is a BFA-sensitive protein on the membranes of Golgi-associated vesicles, and may participate in Golgi-associated vesicles budding and/or sorting, together with actin microfilaments.

Discrimination between Virulent and Attenuated Isolates of Tomato mosaic virus by Restriction Fragment Length Polymorphism

The three missense mutations on the gene for the 130-K protein of Tomato mosaic virus (ToMV) L11A have been thought to be responsible for the attenuation of its virulence. The Eco47I RFLP detecting the missense mutation at 2349 successfully discriminated L11A and its derivative attenuated isolates from ToMV virulent ones. RFLP analysis and mismatch amplification assay detecting the missense mutations at 1117 and 2754, respectively, could not discriminate some of the attenuated isolates from the virulent ones. These results indicated that, of the three missense mutations, only the one at 2349 was conserved in all the L11A-derivative attenuated isolates.

Baseline Cytosolic Ca2+ Oscillations Derived from a Non-endoplasmic Reticulum Ca2+Store

Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.

Outcome of endoscopic intervention for GI bleeding in patients with moderate to severe thrombocytopenia

To study the processing of putative VPg precursors the expression of specific mutant transcripts derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA was examined in a rabbit reticulocyte lysate system. This study revealed that the 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed by mainly intramolecular cleavages at three different sites into three sets of proteins of 60K + 110K, 84K + 87K, and 58K + 112K. Further cleavage of the 60K protein into 58K and VPg has not been observed in this in vitro system. The 84K protein can be further processed by an intramolecular cleavage reaction via two alternative pathways, either into 26K (VPg + 24K) and 58K proteins or into 24K and 60K proteins. VPg can be released from the 112K (VPg + 110K) precursor either directly or via the 26K intermediate. Immunoblot analysis showed that the 112K protein is present in CPMV-infected plant cells indicating that the in vitro observations may hold true in vivo.

Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications.

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K-]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1-,100K-]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K-]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K-]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1-,100K-]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K-]Ad vectors suggest that they may have utility in a variety of gene therapy applications.

Detection and subcellular localization of the turnip yellow mosaic virus 66K replication protein in infected cells.

Turnip yellow mosaic virus (TYMV) encodes a 206-kDa (206K) polyprotein with domains of methyltransferase, proteinase, NTPase/helicase, and RNA-dependent RNA polymerase (RdRp). In vitro, the 206K protein has been shown to undergo proteolytic processing, giving rise to the synthesis of 140-kDa (140K) and 66-kDa (66K) proteins, the latter comprising the RdRp protein domain. Antibodies were raised against the 66K protein and were used to detect the corresponding viral protein in infected cells; both leaf tissues and protoplasts were examined. The antiserum specifically recognized a protein of approximately 66 kDa, indicating that the cleavage observed in vitro is also functional in vivo. The 66K protein accumulates transiently during protoplast infection and localizes to cellular membrane fractions. Indirect immunofluorescence assays and electron microscopy of immunogold-decorated ultrathin sections of infected leaf tissue using anti-66K-specific antibody revealed labeling of membrane vesicles located at the chloroplast envelope.

The open reading frame 1-encoded ('36K') protein of Carnation Italian ringspot virus localizes to mitochondria.

The localization of the 36 kDa ('36K') protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K-GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

Human Immunodeficiency Virus Type 1 VPU Protein Affects Sindbis Virus Glycoprotein Processing and Enhances Membrane Permeabilization

The human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that forms oligomeric structures in membranes. Expression of vpu using Sindbis virus (SV) as a vector leads to permeabilization of plasma membrane to hydrophilic molecules and impaired maturation of wild type SV glycoproteins in BHK cells. The 6K protein is a membrane protein encoded in the SV genome that facilitates budding of virus particles and regulates transport of viral glycoproteins through the secretory pathway. Some of these functions were assayed with a SV mutant containing a partially deleted 6K gene. Transfection of BHK cells with pSVΔ6K vector rendered defective SVΔ6K virus, which had lower membrane permeabilization, impaired glycoprotein processing, and deficient virion budding. Replacement of 6K function by HIV-1 Vpu in SVΔ6K was tested by cloning the vpu gene under a duplicated late promoter (pSVΔ6KVpu). The presence of the vpu gene in the 6K-deleted virus enhances membrane permeability, modifies glycoprotein precursor processing, and facilitates infectious virus particle production. Restoration of infectivity of 6K-deleted SV by Vpu was evidenced by increased PFU production and cytopathic effect on infected cells. The modification of SVΔ6K glycoprotein maturation by Vpu was reflected in augmented processing of B precursor and impairment of PE2 cleavage. Taken together, our data support the notion that HIV-1 Vpu and SV 6K proteins share some analogous functions.

A protochlorophyllide-dependent protein import complex of chloroplasts

Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide, which accumulate in light- and dark-grown plants, respectively. Protochlorophyllide is bound to two NADPH:Pchlide oxidoreductases (POR), termed PORA and PORB, which in angiosperms form higher molecular weight complexes in the prolamellar body of etioplasts. Both POR proteins are nuclear gene products which must be imported from the cytosol.. Import of the precursor of the PORA requires Pchlide and is due to the operation of a second, substrate-dependent protein import site in the plastid envelope. Here we report the isolation of components of the Pchlide-dependent import (PIM) complex and show that it is composed of at least 12 different polypeptides. Among them is a Pchlide a oxygenase which provides Pchlide b as the import substrate of the pPORA, and a tyrosine amino transferase presumably involved in conferring photoprotection onto the PIM complex. Two other constituents were found to be related to previously characterized outer envelope membrane proteins: a 33K protein to TOC33 which is a GTP binding protein, and a 16K protein to a voltage-dependent, amino acid-sective channel protein in the outer plastid envelope.

The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity.

The localization of various Ca(2+) transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca(2+) waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca(2+) signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca(2+) signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate betaCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP(3)R3), and the Gbetagamma subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca(2+) pump, the G protein subunits Galphaq and Gbetagamma, the beta1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various signaling and Ca(2+) transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca(2+) signaling proteins. Hence, the interaction between the Sec6/8 and Ca(2+) signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca(2+) signaling at a step upstream of Ca(2+) release by IP(3). Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca(2+) waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca(2+) signaling complexes and regulation of their activity.

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.

Norwalk virus open reading frame 3 encodes a minor structural protein.

Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3' end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3' end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

Adenovirus-specific translation by displacement of kinase Mnk1 from cap-initiation complex eIF4F.

Translation of cellular mRNAs involves formation of a cap-binding translation initiation complex known as eIF4F, containing phosphorylated cap-binding protein eIF4E, eIF4E kinase Mnk1, eIF4A, poly(A)-binding protein and eIF4G. Adenovirus is shown to prevent cellular translation by displacing Mnk1 from eIF4F, thereby blocking phosphorylation of eIF4E. Over expression of an eIF4E mutant that cannot be phosphorylated by Mnk1 impairs translation of cellular but not viral late mRNAs. Adenovirus 100k protein is shown to bind the C-terminus of eIF4G in vivo and in vitro, the same region bound by Mnk1. In vivo, 100k protein displaces Mnk1 from eIF4G during adenovirus infection, or in transfected cells. Purified 100k protein also evicts Mnk1 from isolated eIF4F complexes in vitro. A mutant adenovirus with a temperature-sensitive 100k protein that cannot inhibit cellular protein synthesis at restrictive temperature no longer blocks Mnk1 binding to eIF4G, or phosphorylation of eIF4E. We describe a mechanism whereby adenovirus selectively inhibits the translation of cellular but not viral mRNAs by displacement of Mnk1 from eIF4G and inhibition of eIF4E phosphorylation.

Identification and Characterization of a 30K Protein (Ad4E3-30K) Encoded by the E3 Region of Human Adenovirus Type 4

Human adenovirus type 4 (Ad4), the sole member of subgroup E, contains an open reading frame in the E3 region predicted to encode a unique 30-kDa protein (named Ad4E3-30K). Ad4E3-30K is predicted to be an integral membrane protein containing an N-terminal signal sequence, a lumenal domain, a transmembrane domain near the C-terminus, and a 37-amino-acid cytoplasmic tail. To determine whether this protein is expressed, rabbit polyclonal antisera were raised against 30K-containing fusion proteins expressed in bacteria. A 30K protein was detected by immunoprecipitation from cell-free translation products and from Ad4-infected A549 cells radiolabeled in the presence of tunicamycin. The protein was detected at only low levels in infected cells. It was not synthesized by a mutant with a large E3 deletion that includes the Ad4E3-30K gene. This mutant grows as well as wild-type Ad4 in culture. Features of Ad4E3-30K were characterized in different transient expression systems. The protein underwent glycosylation by addition of approximately six asparagine-linked oligosaccharides. These glycans were sensitive to endoglycosidase H, indicating that they were either high-mannose or hybrid types, but not complex types, and that the protein did not pass through the Golgi apparatus. Immunofluorescence staining of transfected cells revealed that Ad4E3-30K was localized primarily in the endoplasmic reticulum and nuclear envelope.