The open reading frame I (ORF I)/ORF II part of the human T-cell leukemia virus type I X region is dispensable for p40tax, p27rex, or envelope expression.

The X region of the human T-cell leukemia virus type I contains the second coding exon of the tax and rex regulatory proteins (open reading frame IV [ORF IV] and ORF III, respectively), as well as coding regions for more recently described proteins, p30II (or the tof protein) and p13II in ORF II and the putative rof protein and p12I in ORF I. Deletions and transcomplementation experiments showed that expression of the envelope, as well as that of the tax and rex proteins, was independent of the proteins encoded in the ORF I/ORF II region. Furthermore, p30II and p12I proteins could not replace the rex protein in a rex-dependent envelope or Gag protein expression system.     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.     

Endoplasmic Reticulum and cis-Golgi Localization of Human T-Lymphotropic Virus Type 1 p12I: Association with Calreticulin and Calnexin

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12(I), in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12(I) may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12(I) in cell activation, we characterized the subcellular localization of p12(I) in transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12(I) accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The location of p12(I) was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12(I) with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12(I) directly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12(I) in the establishment of HTLV-1 infection by activation of host cells.     

Proteomics analysis of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus identified two new occlusion-derived virus-associated proteins, HA44 and HA100

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.     

C-terminal region of human T cell lymphotropic virus type I (HTLVI) p19 core protein is immunogenic in humans and contains an HTLVI-specific epitope

To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLVI p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLVI+ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gag-encoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLVII or HTLVIII proteins or with HTLVIII-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLVI-infected patients and is HTLVI specific.     

Immunochemical studies of Lolium perenne (rye grass) pollen allergens, Lol p I, II, and III

It was reported earlier that human immune responses to three perennial rye grass (Lolium perenne) pollen allergens, Lol p I, II, and III, are associated with histocompatibility leukocyte antigen (HLA)-DR3. Rye-allergic people are often concordantly sensitive to all three of these allergens. Since earlier studies suggested that these antigens are non-cross-reactive, their immunologic relatedness by double antibody radioimmunoassay (DARIA) was studied in order to understand further the immunochemical basis for the concordant recognition of the three allergens. Direct binding DARIA studies were performed with human sera from 189 allergic subjects. Inhibition DARIA studies were carried out with 17 human sera from grass-allergic patients who were on grass immunotherapy, one goat anti-serum, and six rabbit antisera. None of the sera detected any significant degree of two-way cross-reactivity between Lol p I and II, or between Lol p I and III. However, the degree of two-way cross-reactivity between Lol p II and III exhibited by individual human and animal antisera varied between undetectable and 100%. In general, the degree of cross-reactivity between Lol p II and III was higher among human sera than among animal sera. Taken together with earlier findings that antibody responses to Lol p I, II and III are associated with HLA-HDR3, and that most Lol p II and III responders are also Lol p I responders, but not vice versa, our present results suggest the following: the HLA-DR3-encoded Ia molecule recognizes a similar immunodominant Ia recognition site (agretope) shared between Lol p I and Lol p II and/or III; in addition, Lol p I appears to contain unique Ia recognition site(s) not present in Lol p II and III. However, further epitope analyses are required to investigate these possibilities.     

The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase.

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.     

Localization of mitochondrial DNA encoded cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments.     

A novel subfamily of monomeric inorganic pyrophosphatases in photosynthetic eukaryotes

Two sPPases (soluble inorganic pyrophosphatases, EC 3.6.1.1) have been isolated from the microalga Chlamydomonas reinhardtii. Both are monomeric proteins of organellar localization, the chloroplastic sPPase I [Cr (Ch. reinhardtii)-sPPase I, 30 kDa] is a major isoform and slightly larger protein than the mitochondrial sPPase II (Cr-sPPase II, 24 kDa). They are members of sPPase family I and are encoded by two different cDNAs, as demonstrated by peptide mass fingerprint analysis. Molecular phylogenetic analyses indicated that Cr-sPPase I is closely related to other eukaryotic sPPases, whereas Cr-sPPase II resembles its prokaryotic counterparts. Chloroplastic sPPase I may have replaced a cyanobacterial ancestor very early during plastid evolution. Cr-sPPase II orthologues are found in members of the green photosynthetic lineage, but not in animals or fungi. These two sPPases from photosynthetic eukaryotes are novel monomeric family I sPPases with different molecular phylogenies and cellular localizations.     

Assortment of phosphatidylinositol 3-kinase complexes--Atg14p directs association of complex I to the pre-autophagosomal structure in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.     

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells to Ras-mediated apoptosis

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.     

Alternatively spliced exon 5 of the FERM domain of protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

Direct physical linkage of MAGUKs to the actin cytoskeleton was first established by the interaction of erythrocyte p55 with the FERM domain of protein 4.1R. Subsequently, it was reported that p55 binds to a 51-amino acid peptide, encoded by exon 10, located within the FERM domain of protein 4.1R. In this study, we investigated the nature of the p55-FERM domain binding interface and show that p55 binds to a second 35-amino acid peptide, encoded by an alternatively spliced exon 5, located within the FERM domain of protein 4.1R. Competition and Surface Plasmon Resonance-binding measurements suggest that the peptides encoded by exons 5 and 10 bind to independent sites within the D5 domain of p55. Interestingly, the full length 135 kDa isoform of protein 4.1R containing both exons 5 and 10 was targeted exclusively to the plasma membrane of epithelial cells whereas the same isoform without exon 5 completely lost its membrane localization capacity. Together, these results indicate that p55 binds to two distinct sites within the FERM domain, and the alternatively spliced exon 5 is necessary for the membrane targeting of protein 4.1R in epithelial cells. Since sequences similar to the exon 5-peptide of protein 4.1R and D5 domain of p55 are conserved in many proteins, our findings suggest that a similar mechanism may govern the membrane targeting of other FERM domain containing proteins.     

Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies.

The 7.5-kb plus-stranded genomic RNA of rabbit hemorrhagic disease virus contains two open reading frames of 7 kb (ORF1) and 351 nucleotides (ORF2) that cover nearly 99% of the genome. The aim of the present study was to identify the proteins encoded in these open reading frames. To this end, a panel of region-specific antisera was generated by immunization of rabbits with bacterially expressed fusion proteins that encompass in total 95% of the ORF1 polyprotein and almost the complete ORF2 polypeptide. The antisera were used to analyze the in vitro translation products of purified virion RNA of rabbit hemorrhagic disease virus. Our studies show that the N-terminal half of the ORF1 polyprotein is proteolytically cleaved to yield three nonstructural proteins of 16, 23, and 37 kDa (p16, p23, and p37, respectively). In addition, a cleavage product of 41 kDa which is composed of VPg and a putative nonstructural protein of approximately 30 kDa was identified. Together with the results of previous studies which identified a trypsin-like cysteine protease (TCP) of 15 kDa, a putative RNA polymerase (pol) of 58 kDa, and the major capsid protein VP60, our data establish the following gene order in ORF1: NH2-p16-p23-p37 (helicase)-p30-VPg-TCP-pol-VP60-COOH. Immunoblot analyses showed that a minor structural protein of 10 kDa is encoded in ORF2. The data provide the first complete genetic map of a calicivirus. The map reveals a remarkable similarity between caliciviruses and picornaviruses with regard to the number and order of the genes that encode the nonstructural proteins.     

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.