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PubMed1995

Human T helper cells specific for HIV reverse transcriptase: possible role in intrastructural help for HIV envelope-specific antibodies.

Cooperation between B cells specific for an antigen exposed on a viral structure and T helper (Th) cells specific for an internal antigen, as demonstrated with influenza, hepatitis B and rabies viruses, has been termed intrastructural help. Th cells specific for internal proteins of HIV, which are much less mutated than its exposed antigens, may be valuable in vaccine design against this virus. We investigated the human Th repertoire specific for the core HIV antigen reverse transcriptase (p66), and determined whether these cells could be candidate intrastructural T helpers. CD4+ T lines and clones were generated from non-immune individuals by stimulation with p66-pulsed antigen-presenting cells (APC). Specific lines were obtained with p66 from 19 out of 21 (90%) of these individuals, vs. 7 out of 29 (24%) with gp120. Diverse epitopes were recognized by different individuals, and various V beta genes were used by these clones. Clones using the same V beta genes were of diverse origin, according to VDJ region sequence. Of these lines 45% responded to p66 in the context of HIV virions. Moreover, p66-specific clones could respond to APC that had internalized HIV complexed with envelope-specific monoclonal antibodies, suggesting that p66-specific Th cells may participate in intrastructural help. These studies indicate that p66-specific Th cells are detectable in vitro in most naive individuals and exhibit clonal heterogeneity, and that the majority recognize an HIV conserved antigen. They respond to p66 following processing of whole virions and are clearly candidates for intrastructural help. If confirmed in vivo, p66 should be included among vaccine candidates investigated to optimize the anti-HIV Th response.

Amino Acid Sequence↗

PubMed1980

Changes in tumor-specific antigen expression during passage in vitro and in vivo of newly derived methylcholanthrene-induced sarcomas of BALB/C mice.

The characteristics of tumor antigens of the transplantation type, TATA, were studied in a series of methylcholanthrene-induced sarcomas in pedigreed BALB/cAN mice. Each of the six sarcomas exhibited unique TATA when assayed for tumor rejection in syngeneic hosts. In three instances, however, in sarcomas CI-I, CII-5 and CII-7, TATA activity was lost during early in vivo passages. This activity was reestablished invivo from the in vitro-passaged lines in CI-I and CII-7 and these sarcomas along with CI-3, CI-4 and CII-10 maintained a stable TATA phenotype throughout the 50 transplant passages; TATA was lost permanently, however, in CII-5. These results indicate a dimorphic nature of early-passage neoplasms. No cross-reacting antigens were detected among these sarcomas, including also Meth A, a sarcoma well characterized as to its membrane antigens; nor was any evidence obtained for the existence of alien (inappropriate) H-2 antigens employing tumor rejection assays in F1 hybrids of BALB/c with strains bearing several haplotypes and also with studies of H-2 serology. In three sarcomas, some evidence was obtained for the existence of alloantigens of the non-H-2 type since it was found that (BALB/c X DBA/2)F1 hybrids could not be immunized by respective sarcomas. These findings suggest the existence on these sarcomas of a tumor antigen that is expressed normally in DBA/2 mice, although no definitive evidence has been obtained. Assays for MuLV, for the viral structural proteins gp70 and p30 and for reverse transcriptase showed that three sarcomas were positive and three others, as well as Meth A, were negative. MuLV and its antigens had no influence on tumor rejection activity nor could the cross-reactivity observed in the radioisotopic footpad assay (IFP) be related to MuLV.

Animals↗

PubMed1998

Recombination of nonreplicative RNA precursors of Sindbis virus in infected cells overexpressing murine-inducible nitric oxide synthase.

The Sindbis virus-based SINrep5 expression system is one of the most efficient vectors for gene transfer leading to fast and high expression of the gene of interest. This system was used to transfect vascular endothelial and smooth muscle cells using murine inducible nitric oxide synthase (miNOS) as a reporter gene. Infection of both cell types leads to high expression levels of miNOS. In addition, the harvested supernatant of these infected cells was used for further rounds of infections, demonstrating that recombination of the parental RNA with the helper RNA takes place and results in the production of infectious particles. As shown by RT-PCR, after recombination the miNOS gene is located in between the nonstructural and structural viral genes. This study demonstrates that despite claims in other publications, the Sindbis virus-based SINrep5 expression system leads to recombination and is thus not a safe system for in vitro and in vivo applications.

Animals↗

PubMed1998

HIV-1 rev nuclear export signal binding peptides isolated by phage display.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is absolutely essential in the viral replication cycle, where it induces the production of viral structural proteins. Rev functions in part by inducing the nuclear export of incompletely spliced mRNA species specified by the presence of an RNA element, the Rev response element (RRE). Several proteins implicated in RNA processing and nucleo-cytoplasmic transport have been shown to interact with Rev, however, their exact roles remain unknown. To map potential protein recognition sites within the Rev structure, we have screened a phage library, displaying random 15-mer peptides, and isolated clones exhibiting similar sequences that specifically interact with Rev. The binding sites on Rev of the corresponding synthetic peptides were characterised by protein footprinting, involving partial proteolysis of radioactively end-labelled Rev protein. Two of the peptides produced a significant footprint within the nuclear export signal of Rev, raising the possibility that they mimic the binding of cellular protein factors implicated in nuclear export.

Amino Acid Sequence↗

PubMedDate not supplied

Three-dimensional reconstruction of icosahedral particles--the uncommon line.

The past few years have seen an explosion in the number of viral structures determined by icosahedral reconstruction from cryoelectron micrographs. The success of this work has depended upon a combination of the high-fidelity but low-contrast information contained in these images with efficient algorithms for determining particle orientation and three-dimensional structure. This review describes the principles behind the most commonly used method of reconstruction of the icosahedral particles and the method's implementation in an icosahedral reconstruction program suite.

Algorithms↗

PubMed1999

Transmission of human immunodeficiency type 1 viruses with intersubtype recombinant long terminal repeat sequences.

Retroviruses such as human immunodeficiency virus type 1 (HIV-1) contain two RNA strands per virion, and recombination can occur frequently during reverse transcription. Recombination may occur between HIV-1 genomes of the same subtype or among genomes of two or more distinct subtypes present in an individual. In the current study, we found that recombinatorial events were not limited to viral structural genes such as gag and env, but rather, recombination could likewise occur within the 5' long terminal repeat (LTR). Intersubtype recombinant LTRs among HIV-1 subtypes A, C, and D were found in Tanzanian infants. By introducing novel LTR sequences, these recombinant LTR viruses may further increase the adaptive potential and fitness of HIV-1.

Base Sequence↗

PubMed2006

Immunity and autoimmunity induced by polyomaviruses: clinical, experimental and theoretical aspects.

In this chapter, polyomaviruses will be presented in an immunological context. Principal observations will be discussed to elucidate humoral and cellular immune responses to different species of the polyomaviruses and to individual viral structural and regulatory proteins. The role of immune responses towards the viruses or their proteins in context of protection against polyomavirus induced tumors will be described. One central aspect of this presentation is the ability of polyomaviruses, and particularly large T-antigen, to terminate immunological tolerance to nucleosomes, DNA and histones. Thus, in the present chapter we will focus on clinical, experimental and theoretical aspects of the immunity to polyomaviruses.

Autoimmunity↗

PubMed1995

Active sites of ligands and their receptors are made of common peptides that are also found elsewhere.

The simultaneous emergence in evolution of a ligand and its receptor might have entailed their active sites being drawn from the pool of common oligopeptides. This was tested on the principal components of cell-matrix interaction: the RGD (Arg-Gly-Asp) site of matrix proteins and the EKKD (Gly-Lys-Lys-Asp) site of integrin cell-surface receptor. In the 32 diverse proteins scrutinized, which totalled 14,806 residues, there were 104 Arg-Gly dipeptides. Most common of the tripeptides beginning with Arg-Gly were Arg-Gly-Leu, Arg-Gly-Gly, and Arg-Gly-Asp; each was found in ten copies. RGD tripeptide was one of the commonest; the fortuitous presence of an RGD site was noted in two enzymes, fibrinogen, a pituitary hormone precursor, and a viral structural protein. The 32 proteins also contained 121 Lys-Lys dipeptides. Of the tetrapeptides centered by Lys-Lys, the commonest was Lys-Lys-Lys-Lys, in four copies. Second most common were Gly-Lys-Lys-Lys, Val-Lys-Lys-Leu, and Glu-Lys-Lys-Asp; each occurred in three copies. The fortuitous presence of an EKKD site was noted in three proteins--an intracellular transport protein, a pituitary hormone precursor and a protein of the cerebrospinal fluid. In most instances, protein-protein interaction between the fortuitously present active sites appears to bring about deleterious consequences. Occasionally, however, the fortuitous active site appears to confer a new function to a protein bearing it.

Amino Acid Sequence↗

PubMed1995

Molecular evidence for the existence of disseminated zoster as a distinct entity in an immunosuppressed renal transplant patient.

Immunosuppressed renal transplant recipient are at substantially increased risk for the development of varicella zoster virus infections. They are also more prone than immunocompetent patients to develop atypical zoster and to experience a protracted course, and among them there is a higher frequency of generalized infections with possible fatal outcome. While establishing the diagnosis is essential to provide adequate therapy, conventional laboratory methods frequently fail to confirm the suspected infection. We report on a 47-year-old renal transplant recipient who developed multiple necrotic cutaneous ulcers under immunosuppressive treatment. While electron-microscopic analysis (negative staining) revealed no viral structures, varicella zoster virus specific DNA was detected by polymerase chain reaction in material obtained by a swab from these ulcers. Atypical herpetic infection should also be considered as a cause of disseminated ulcerative or necrotic skin lesions in immunosuppressed patients. Assays based on polymerase chain reaction are useful for the rapid confirmation or rejection of the suspected diagnosis of atypical herpetic infection.

Base Sequence↗

PubMed1976

[The neuronal Torres bodies in experimental yellow fever encephalomyelitis. An additional example of the heterogeneous nature of Cowdry type A intranuclear inclusions].

In experimental yellow fever virus encephalomyelitis of adult albino mice, the occurrence of intranuclear inclusions within nerve cells, in particular, within spinal and bulbar motoneurons, is a constant finding. The light microscopic features of these so-called neuronal Torres bodies correspond entirely to the criteria laid down by Cowdry for the type A intranuclear inclusions. At the ultrastructural level, these karyoplasmic formations consist of haphazardly distributed patches of an essentially amorphous material of various density. Unlike the Cowdry type A intranuclear inclusions characteristic of several herpesvirus and paramyxovirus infections, neuronal Torr-s bodies do not contain any viral structures. The frequency with which neuronal Torres bodies, certain nucleolar alterations and severe tigrolytic changes occur together, possibly indicates that this sort of type A intranuclear inclusion developes as a consequence of a virus induced disturbance of the cell nucleic acid and/or protein anabolism.

Animals↗

PubMed1994

Mechanisms of action of NIP71 on N-myristoyltransferase activity.

N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT.II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (microliter) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence of myristoylated alanine-rich C-kinase substrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP71) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291:635-639, 1993) potently inhibited highly purified NMT activity (IC50 23.7 nM). A minor NMT activity (NMT.PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP71 inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP71 having an apparent higher affinity for NMT than the NMT.myristoyl.CoA complex. Inhibition by NIP71 at subsaturating concentrations of myristoyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.

Acyltransferases↗

PubMed1991

Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies.

We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency. The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay.

Animals↗

PubMed1986

Detection of retroviral particles in hybridomas secreting monoclonal antibodies.

Electronmicroscopy of hybridoma clones derived by fusing BALB/c mouse spleen cells with P3U1 mouse plasmacytoma cells to generate monoclonal antibodies against human sarcoma antigens, revealed the presence of large number of viral particles. These particles were also seen budding from the cell surfaces. The intracytoplasmic particles were intracisternal and resembled type-A oncornavirus, while the budding and extracellular forms, with a centrally located nucleoid, resembled mature type-C oncornaviruses. Cells of the parental P3U1 palsmacytoma cell line and of the NS-1 myeloma cell line contained morphologically identical viral structures. The scientific and medical communities engaged in hybridoma research should be alert to the possible presence of viruses in hybridomas and their products. The question is raised as to whether it is safe to use mouse monoclonal antibodies for clinical purposes, both diagnostic and therapeutic.

Animals↗

PubMed1998

[Prospects of gene therapy in treatment of coronary heart disease].

Gene therapy describes the transfer of genetic material into a cell for therapeutic purposes. This opens new therapeutic perspectives for cardiovascular medicine. It includes the inhibition of restenosis following angioplasty, e.g., by transfer of suicide genes such as CMV-thymidine kinase or by inhibition of the cell cycle of cells within the vessel wall. On the other hand, there is promising data concerning the induction of therapeutic angiogenesis using the transfer of angiogenic genes such as the one for vascular endothelial growth factor VEGF. During the past three years significant progress was made by a number of preclinical studies. On the other hand, the therapeutic success of gene therapy in humans is still missing, and this is true for all different strategies tested so far. Important and basic issues of gene transfer and the resulting cellular response need to be solved before a therapeutic use might become a routine procedure. In the meanwhile, an important focus of the experimental work lies in the identification and characterization of molecular targets for therapeutic interventions, another in the improvement of gene transfer systems. Such work will provide new information about the biology of cellular and viral structures including their functional interrelation; in addition a better insight into the pathogenesis of the various disease processes should be obtained.

Animals↗

PubMed2005

Generation of recombinant norovirus-like particles (VLP) in the human endothelial kidney cell line 293T.

Noroviruses (NVs) are the major cause of non-bacterial outbreaks of gastroenteritis. Here we report a new alternative system to generate recombinant NV virus-like particles (rNV-VLP) in a human endothelial kidney cell line (HEK). Transfecting HEK-293T cells with an expression vector coding for the ORF-2 gene lead to the expression of the viral structural protein VP1 which spontaneously assembled into virus-like particles (VLP), as shown by electron microscopy. The transfected cells did not show a cytopathic effect and released rNV-VLP into the culture medium. The HEK-293T cell derived particles were morphologically indistinguishable to the rNV-VLP produced from baculovirus and the Venezuelan equine encephalitis virus (VEE)-replicon. The produced particles were stable for at least 2.5 months at 4 degrees C.

Cell Line↗

PubMed1989

Estrogen influence on maturational pathway of murine mammary tumor virus: an immunoelectron microscopy study.

Modifications induced by estrogens on hormone-independent murine mammary tumor (MMT) and its main etiological agent, the MMT virus (MMTV), are reported. High doses of estrogens released continuously from silastic capsules delay significantly the development of transplanted tumors into syngeneic hosts. Neoplastic cells present a striking cytoplasmic vacuolization and changes in the MMTV differentiation pattern. Mature virions are detected budding into cytoplasmic vacuoles instead of the extracellular space as in spontaneous and untreated transplanted tumors. This phenomenon is reversed after estrogen withdrawal at the first sign of tumor development. Application of electron microscope immunocytochemistry with colloidal gold-protein A complex and multiple monospecific antibodies reveals several interesting features. In spontaneous and untreated tumor grafts, structural viral proteins p14 and p25 appear in both intracytoplasmic capsids and mature extracellular viruses. By contrast glycoprotein gp55 labels only the envelope of mature virus. In estrogen-treated tumors this antigenic pattern is modified and the gp55 is detected in those atypical virions maturing into the intracytoplasmic vacuoles. These observations led to the conclusions that the delay in the development of hormone-independent mammary tumors caused by estrogen is due to an abnormal maturational viral process and that estrogens induce alterations of polarity in the translocation process of viral envelope glycoproteins.

Animals↗

PubMed1995

Detection of intracellular HIV-1 Rev protein by flow cytometry.

The Rev trans-activator protein plays a pivotal role in human immunodeficiency virus type 1 (HIV-1) replication by allowing expression of the viral structural proteins. We have developed a protocol to quantitatively assay intracellular steady state levels of Rev Ag (Rev wild type and RevM10 proteins) by flow cytometry. Three fixation and permeabilization techniques were compared. These protocols varied in the magnitude of the signal which could be detected, and in the ability to distinguish between Rev Ag positive and negative populations. This technology is applicable to a variety transduced or transfected cell types (species, lineage), and for cell lines and primary cells acutely infected with HIV-1. The assay is therefore a valuable tool both to analyze Rev protein expression levels in HIV-infected cells and to optimize delivery of the dominant-negative RevM10 gene for clinical gene therapy applications. In addition, a second, independent intracellular protein (HIV-Tat) has been detected using the same approach.

3T3 Cells↗

PubMed1992

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni.

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.

Animals↗