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G N Pavlakis

Publications and source records attributed to G N Pavlakis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation.

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.

Adaptor Protein Complex alpha Subunits↗
PubMed1997

Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A.

A chimeric protein consisting of the cholecystokinin receptor type A (CCKAR) and the green fluorescent protein (GFP) was used for studying receptor localization, internalization, and recycling in live cells in real time in four different cell lines. Fusion of the C terminus of the CCKAR to the N terminus of the GFP did not alter receptor ligand binding affinity, signal transduction, or the pattern of receptor surface expression and receptor-mediated cholecystokinin (CCK) internalization. The use of a new GFP mutant with increased fluorescence allowed the continuous observation of CCKAR-GFP in stably expressing cell lines. Newly obtained biologically active fluorescent derivatives of CCK were used for simultaneous observation of receptor and ligand trafficking in CHO, NIH/3T3, and HeLa cells stably expressing the fluorescent CCKAR and in transiently transfected COS-1 cells. Receptor internalization was predominantly ligand dependent in HeLa, COS-1, and CHO cells, but was mostly constitutive in NIH/3T3 cells, suggesting the existence of cell-specific regulation of receptor internalization. The CCKAR antagonists, L-364,718 and CCK 27-32 amide potently inhibited spontaneous internalization of the receptor. The average sorting time of CCK and the receptor in the endosomes was about 25 min. The receptor recycled back to the cell membrane with an average time of 60 min. While the ligands sorted to lysosomes, no receptor molecules could be detected there, and no receptor degradation was observed during recycling. These results demonstrate the usefulness of GFP tagging for real time imaging of G protein-coupled receptor trafficking in living cells and suggest that this technique may be successfully applied to the study of the regulation and trafficking mechanisms of other receptors.

3T3 Cells↗
PubMed1997

The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding.

The inducibly expressed GTPase (IGTP) is representative of a newly identified group of interferon gamma-inducible GTPases, whose functions are currently unknown. We have begun to address the cellular function of IGTP by examining its subcellular distribution and its guanine nucleotide binding status. Using immunofluorescence, electron microscopy, and subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264. 7 macrophages and C127 fibroblasts. In the immunostaining experiments, staining of discrete cytoplasmic structures on the periphery of the endoplasmic reticulum was also evident. Using polyethyleneimine-cellulose thin layer chromatography, the guanine nucleotides that complexed to immunoprecipitated IGTP, in both control and interferon gamma-stimulated cells, were 90-95% GTP and 5-10% GDP, suggesting that the protein was in an active state. A mutant IGTP protein was created that had no detectable complexed GTP, and in both subcellular fractionation and IGTP-green fluorescent protein fusion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is independent of its capacity to localize to the endoplasmic reticulum. Given these results, we propose that IGTP is representative of a new family of endoplasmic reticulum GTPases that may be involved in protein processing or trafficking.

Amino Acid Sequence↗
PubMed1997

Inflammatory polyarthropathy and bone remodeling in HTLV-I Tax-transgenic mice.

Transgenic mice carrying the tax gene of human T-cell lymphotropic virus type I displayed a high prevalence of arthropathy. The percentage of affected animals increased with age, reaching a peak of 43% at 20 months. Southern analysis of deoxyribonucleic acid (DNA) from tissue samples indicated that disease development was related to tax copy number. Histopathologic evaluation of the ankle joints disclosed deep erosion of the synovial lining, fibrous tissue proliferation together with angiogenesis, mononuclear cell infiltration, and activation of osteoclasts. Radiologic examination confirmed joint involvement and revealed bone architecture modifications. The phenotype exhibited by the affected animals closely resembles that of seronegative arthritis in humans, and may indicate tax protein as a causal agent of arthropathy observed in HTLV-I infected individuals.

Age Factors↗
PubMed1997

Preferential binding of poly(A)-binding protein 1 to an inhibitory RNA element in the human immunodeficiency virus type 1 gag mRNA.

Human immunodeficiency virus type 1 (HIV-1) mRNAs encoding structural proteins contain multiple inhibitory/instability elements (INS), which decrease the efficiency of viral protein expression. We have previously identified a strong INS element (INS-1) within the p17(gag) coding region. Here we show that poly(A)-binding protein 1 (PABP1) binds preferentially to INS-1 within the p17(gag) mRNA, but not to a mutated mRNA in which INS-1 function is eliminated. Competition experiments performed in the presence of different nucleic acids and homoribopolymers demonstrated preferential binding of PABP1 to the INS-1-containing RNA. In contrast to HeLa cells and several lymphoid cell lines, certain human glioma cell lines exhibit high levels of gag expression in the absence of Rev upon transient transfection with wild type gag expression vectors. We analyzed extracts of different cell lines and found that the binding of PABP1 to INS-1 RNA is significantly diminished in glial cell extracts. The expression levels of gag correlate with the absence of binding of PABP1 to the INS-1 RNA in cellular extracts. These results suggest a role for PABP1 in the inhibition of gag expression mediated through INS-1.

Blotting, Western↗
PubMed1997

Improved DNA: liposome complexes for increased systemic delivery and gene expression.

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

Animals↗
PubMed1997

Reduced viral load and lack of CD4 depletion in SCID-hu mice infected with Rev-independent clones of human immunodeficiency virus type 1.

The posttranscriptional control element CTE of the simian type D retrovirus has been shown to support replication of Rev-Rev-responsive-element (RRE)-deficient molecular clones of human immunodeficiency virus type 1 (HIV-1). Upon infection of peripheral blood mononuclear cells in vitro, these CTE-containing Rev-independent viruses that are nef+ or nef-minus showed lower replicative capacity and infectivity than the wild-type HIV-1. We studied the effects of Rev-RRE replacement by the CTE on HIV-1 expression with SCID-hu mice. The nef+ and nef-minus Rev-independent viruses established infection with kinetics slower than that of the nef-minus NL4-3. Most importantly, no depletion of CD4-bearing thymocytes was observed after 6 weeks for mice infected with these Rev-independent viruses. This is in contrast to the infection with both wild-type and nef-minus viruses, which led to varying depletion of thymocytes. These data suggest an attenuated phenotype for growth and cytotoxicity of the Rev-independent HIV-1 clones in SCID-hu mice, independent of the presence of Nef. The mutant viruses, which have the essential Rev-RRE regulatory system eliminated, display a distinct phenotype not previously observed with HIV mutant viruses having deletions of accessory genes. Therefore, replacement of the Rev-RRE regulatory axis may generate viruses with altered biological properties in vivo.

Animals↗
PubMed1997

Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation.

The expression of gag, pol, and env of human immunodeficiency virus type 1 (HIV-1) depends on the presence of the viral Rev protein. This dependence is, at least in part, due to the presence of negatively acting sequences (inhibitory or instability elements [INS]) located within unspliced and partially spliced mRNAs. The positive interaction of Rev with the Rev-responsive element in these mRNAs counteracts the negative effects of the inhibitory sequences. Here, we demonstrate that in addition to the previously identified INS1 within p17gag, several other INS elements exist within the gag/pol region of HIV-1. These elements act independently of each other and were eliminated by mutagenesis after the introduction of multiple point mutations not affecting the coding region, leading to constitutive high levels of Gag expression. Expression vectors containing an intact or nearly intact p55gag region allowed the production of immature viral particles in mammalian cells in the absence of any other HIV proteins. The introduction of additional mutations in the protease region allowed efficient production of Gag/protease, which resulted in processing of the Pr55gag precursor and production of mature Gag particles with a lentivirus-like conical-core structure. The elimination of a newly identified INS element within pol and the previously identified CRS located within int was accomplished by the same methodology. Sequence comparisons of the identified inhibitory elements revealed no apparent homologies and demonstrated that these sequences are not splice sites. These results demonstrate that the elimination of INS elements leads to efficient expression of HIV-1 mRNAs in the absence of Rev or any posttranscriptional activating mechanisms.

Amino Acid Sequence↗
PubMed1996

Analysis of human immunodeficiency virus type 1 mRNA splicing patterns during disease progression in peripheral blood mononuclear cells from infected individuals.

HIV-1 produces more than 20 mRNAs encoding the viral proteins. We have used a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) approach to determine HIV-1 transcriptional patterns during the course of viral infection in unstimulated peripheral blood mononuclear cells (PBMCs) from different patients. Several sets of PCR primers, used in parallel reactions, allowed the amplification and specific detection of almost all individual HIV-1 transcripts. We investigated the transcriptional profile in two individuals during primary acute and early chronic infection. In these individuals, HIV-1 mRNA expression was elevated at the first time points examined and declined over time. In addition, we performed a detailed study of HIV-1 expression in several individuals over a minimum of 7 years following seroconversion. We found that long-term asymptomatic individuals had undetectable or low levels of the three classes of HIV-1 transcripts (unspliced, singly spliced, and multiply spliced). Individuals who demonstrated disease progression showed either a general increase in the amount of expression of all transcripts or elevated levels of unspliced transcripts in late-stage disease. The splicing pattern in each patient was conserved over the years and differed among the different individuals. No evidence of major changes in the splicing pattern was found during disease progression within the same individual. Thus, HIV-1 transcriptional patterns are viral strain specific rather than disease stage specific. These results indicate that high-level expression of any class of HIV-1 transcripts is associated with clinical progression. Our analysis also demonstrates the importance of using more than one set of primers to evaluate HIV-1 RNA expression, since virus in patient PBMCs showed sequence heterogeneity in conserved regions.

CD4 Lymphocyte Count↗
PubMed1996

Antiretroviral monotherapy in early stage human immunodeficiency virus disease has no detectable effect on virus load in peripheral blood and lymph nodes.

Initiation of antiretroviral monotherapy early in the course of infection with human immunodeficiency virus may result in a temporary slowing in the rate of disease progression; however, little is known about the virologic effects of early therapy. Virus load was measured in peripheral blood and lymph nodes from 16 antiretroviral-naive patients with a mean CD4 T lymphocyte count of 659 cells/microliter at baseline and after 8 weeks of either no treatment or zidovudine therapy. CD4 T lymphocyte counts and all virologic parameters examined remained unchanged regardless of zidovudine treatment status. Histopathology and virus distribution within lymph nodes remained constant between baseline and week 8 in each patient, indicating that the virologic and histologic parameters examined in a single lymph node are representative of a systemic process. Early antiretroviral monotherapy with zidovudine had no effect on virologic parameters in this group of patients with relatively high CD4 T lymphocyte counts and low measures of virus load at baseline.

Adult↗
PubMed1996

Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site.

Base Sequence↗
PubMed1996

The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function.

It was previously shown that a 240-nucleotide (nt) RNA element (cis-acting transactivation element [CTE]) located between the env gene and the 3' long terminal repeat of simian retrovirus type 1 (SRV-1) can functionally replace posttranscriptional activation directed by Rev and the Rev-responsive element (RRE) when inserted into a Rev- and RRE-deficient molecular clone of human immunodeficiency virus type 1, resulting in efficient virus replication. Here, we analyze the molecular and structural requirements for function of this RNA element. Deletion mutagenesis demonstrated that the core element spans 173 nt. SRV-2 and Mason-Pfizer monkey virus have highly homologous elements, which function similarly when inserted into the Rev/RRE-deficient human immunodeficiency virus type 1. Computer prediction indicated that the core CTEs of all three viruses have similar extensive secondary structures. Mutagenesis of the SRV-1 CTE revealed that both sequence and secondary structure are essential for function. Nuclease probing of the SRV-1 CTE further supported the genetic analysis and confirmed the predicted structural features of the RNA element. Sequence analysis of the 240-nt SRV-1 CTE, after continuous long-term propagation of the Rev-independent viruses, revealed that the genetically defined core element remained unchanged, while regions outside the core CTE underwent deletions or duplications. These data further support our in vitro mutagenesis data and demonstrate the importance of the sequence and structure of the SRV-1 CTE for appropriate function.

Base Sequence↗
PubMed1995

Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm.

Expression of gag/pol and env genes of human immunodeficiency virus requires the viral Rev protein. Mutant Rev proteins, displaying a transdominant phenotype (TDRev), were shown to inhibit Rev function. To investigate the underlying mechanism of this inhibition, the green fluorescent protein (GFP) of Aequorea victoria was fused to Rev and TDRev, which allowed the study of their trafficking and interactions in living human cells. Both Rev-GFP and TDRev-GFP were shown to retain appropriate nucleolar localization and function. Upon actinomycin D treatment, Rev-GFP was transported to the cytoplasm within 1.5 hr, while TDRev, although partially dissociated from the nucleolus, was retained in the nucleus. Coexpression of Rev-GFP and TDRev in the same cell demonstrated that TDRev inhibited the transport of Rev-GFP from the nucleus to the cytoplasm. This inhibition was specific for Rev, since the export of the functionally analogous Rex protein of the human T-cell leukemia virus type I was not inhibited by TDRev. These results indicate that Rev and TDRev form heteromultimers in the nucleolus and that this interaction prevents Rev's export from the nucleus to the cytoplasm. In addition to providing a model for the function of TDRev, these results also demonstrate the successful application of protein fusions to GFP to study localization and trafficking of proteins in living mammalian cells.

Base Sequence↗
PubMed1995

Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue.

Although several immunologic and virologic markers measured in peripheral blood are useful for predicting accelerated progression of human immunodeficiency virus (HIV) disease, their validity for evaluating the response to antiretroviral therapy and their ability to accurately reflect changes in lymphoid organs remain unclear. In the present study, changes in certain virologic markers have been analyzed in peripheral blood and lymphoid tissue during antiretroviral therapy. Sixteen HIV-infected individuals who were receiving antiretroviral therapy with zidovudine for > or = 6 months were randomly assigned either to continue on zidovudine alone or to add didanosine for 8 weeks. Lymph node biopsies were performed at baseline and after 8 weeks. Viral burden (i.e., HIV DNA copies per 10(6) mononuclear cells) and virus replication in mononuclear cells isolated from peripheral blood and lymph node and plasma viremia were determined by semiquantitative polymerase chain reaction assays. Virologic and immunologic markers remained unchanged in peripheral blood and lymph node of patients who continued on zidovudine alone. In contrast, a decrease in virus replication in lymph nodes was observed in four of six patients who added didanosine to their regimen, and this was associated with a decrease in plasma viremia. These results indicate that decreases in plasma viremia detected during antiretroviral therapy reflect downregulation of virus replication in lymphoid tissue.

Adult↗
PubMed1995

Intracellular trafficking of the human immunodeficiency virus type 1 Rev protein: involvement of continued rRNA synthesis in nuclear retention.

We have explored the mechanism directing the intracellular trafficking and nucleolar accumulation of the human immunodeficiency virus type 1 (HIV-1) Rev protein. Treatment of Rev-expressing cells with mycophenolic acid, an inhibitor of inosine monophosphate dehydrogenase, resulted in a redistribution of Rev from the nucleoli to the nucleoplasm and cytoplasm. In contrast, a Rev effector domain mutant was retained in the nucleus, indicating the involvement of this domain in the protein's nuclear retention/nucleocytoplasmic transport. Identical results were obtained by inhibiting transcription using actinomycin D or 5,6-dichlorobenzimidazole riboside. All three drugs were found to inhibit biosynthetic labeling of ribosomal RNA and to disrupt nucleolar morphology, suggesting a correlation between nucleolar/nuclear retention of Rev, continued ribosomal RNA synthesis, and intact nucleolar architecture. Results of binding/immunofluorescence assays using isolated, permeabilized nuclei and extracts of cells expressing Rev demonstrated that the protein is able to bind to nucleoli in vitro, in the absence of active cellular processes or eukaryotic posttranslational modifications. Rev derived from actinomycin D-treated cells showed equivalent binding, indicating that the inhibitor did not directly interfere with the ability of the protein to interact with nucleolar structures. Rev's interaction with nucleoli was directed by the protein's arginine-rich RNA-binding/nucleolar localization domain, and was abrogated by pretreatment of the nuclei with RNaseA, indicating a requirement for RNA, probably ribosomal RNA.

Cell Nucleolus↗
PubMed1995

Restriction of human immunodeficiency virus type 1 production in a human astrocytoma cell line is associated with a cellular block in Rev function.

Chronically human immunodeficiency virus type 1 (HIV-1) strain IIIB-infected human TH4-7-5 astrocytoma cells show low-level virus production. Cocultivation of TH4-7-5 cells with myelomonocytic cells led to active virus production in these target cells after a lag period, indicating cell-determined restriction of virus replication in the glial cells. HIV-1 transcript patterns of TH4-7-5 cells contained only a small proportion of Rev-dependent mRNA species, mimicking a Rev-negative phenotype despite the presence of rev mRNAs and protein. Sequencing of the single provirus integrated in TH4-7-5 cells demonstrated that the rev gene and the Rev-responsive element are intact. These results suggested inhibited function of the Rev-regulatory unit in these astrocytoma cells. Transfection of TH4-7-5 cells with a Rev expression plasmid resulted in weak or no induction of proviral p24gag antigen levels compared with the dramatic increase observed in Rev-permissive HeLa cells. Immunofluorescence analysis of TH4-7-5 cells transfected with a rev-expressing plasmid revealed prominent cytoplasmic and nuclear-nucleolar localization of Rev, in contrast to the predominant nuclear-nucleolar localization pattern of Rev in HeLa cells. We conclude that restriction of virus production in TH4-7-5 cells is at least partially due to a block in Rev-dependent posttranscriptional regulation of HIV expression.

Amino Acid Sequence↗
PubMed1995

Efficient expression of the human papillomavirus type 16 L1 protein in epithelial cells by using Rev and the Rev-responsive element of human immunodeficiency virus or the cis-acting transactivation element of simian retrovirus type 1.

Production of the human papillomavirus (HPV) late gene products L1 and L2 is limited to terminally differentiated keratinocytes. Here, we demonstrate that mRNA encoding the HPV-16 L1 capsid protein contains cis-acting RNA elements that inhibit expression at the posttranscriptional level. While cytoplasmic L1 mRNA is detectable in transfected HeLa cells, L1 protein is not produced. We have identified at least one major inhibitory element that is located within the L1 open reading frame, whereas another negative element had been reported to lie in the 3'-untranslated region of L1. The presence of these elements may explain the lack of HPV late gene expression in undifferentiated epithelial cells. Efficient production of HPV-16 L1 could be achieved with posttranscriptional regulatory elements of human immunodeficiency virus type 1 or simian retrovirus type 1. L1 protein was expressed in the presence of human immunodeficiency virus type 1 Rev from hybrid mRNAs containing the RNA binding site for Rev (Rev-responsive element). In addition, we have achieved efficient expression of L1 from hybrid mRNAs containing a cis-acting transactivation element from simian retrovirus type 1. Our data show that HPV-16 L1 protein production is regulated posttranscriptionally. This regulated expression may allow virus production in terminally differentiated epithelial cells and is probably a conserved and important mechanism for HPV expression.

Base Sequence↗