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A virus derived from cells of a Iymphoblastoid line originating from the lymph node of a healthy African green monkey was characterized as a typical member of the foamy virus subgroup of rctroviridac by its morphological, physicochemical, biological and biochemical properties (reverse transcriptase actvity). Besides the usual host range of foamy viruses, the isolated strain revealed a remarkable T -lymphotropism, distinguishing it from the prototypes of foamy viruses previously isolated from African green monkeys. Two foamy virus infectious are demonstrated in human contacts of the African green monkey colony, with the animal barbauring the isolate.
Murine spienie T lymphocytes display maximal cellular myc gene (c-myc) expression already 3 h after concanavalin A timulation and sub equent down-regulation before the onset of DNA syntbesis. Stimulation by leucoagglulinin in the prcsence or absence of interleukin 2 Ieads to only low initiaJ Ievels of c-myc-specific RNA which, however, increase later on. A similar pattero of c-myc expression is shown by the Lyt- 2+ T cell subpopulation stimuiated with eilher concanavalin A or leucoagglutinin in the prescncc of interleukin 2. Although eH]thyn1idine incorporation was identical, the leucoagglutinin-stimulated Lyt-2+ T cells werc void of any demon. trable c-mycspeci. fic RNA at 3 h post-stimulation. Thus, the kinetics of c-myc expression in mause T lymphocytes arenot at all uniform, but depend on the mitogen and the subpopulation. [n contrast, lcvel8 of c-rasH•-spccific R A wcre always low at early times, always increased towards tbe onset ofDNA synthesis and down-regulationwas not observed.
DNA synthesis and adenosine(S')tetraphosphate(S ')adenosine (Ap.A) levels decrease in cells treated with EDTA. The inhibitory effect of EDTA can be reversed with micro molar amounts of ZnCI2• ZnCh in micromolar concentrations also inhibits Ap.A hydrolase and stimulates amino acid-dependent Ap.A synthesis, suggesting that Zn2+ is modulating intracellular Ap.A pools. Serum addition to GI-arrested cells enhances uptake of Zn, whereas serum depletion leads to a fivefold decrease of the rates of zinc uptake. These results are discussed by regarding Zn2+ as a putative 'second messenger' of mitogenic induction and Ap.A as a possible 'third messenger' and trigger of DNA synthesis.
Newly isolated lymphocytes from mousespleensexpress the c-fos oncogene even in the absence of mitogen with maximal mRNA levels 60 min post preparation of single cell suspension, whereas c-myc mRNA Ievels increase only after mitogenic Stimulation with maximal mRNA Ievels 6 h post Stimulation. The half-lives of c-fos mRNA are generally very short; they increase from 14 min (after 30 min of culture) to 70 min (after 2 h of culture). The half-lives of c-myc mRNA decrease from 50 min (at 2 and 6 h post stimulation with concanavalin A) to 12 min (at 48 h post stimulation). The c-fos gene transcription is already tumed on in time-0 lymphocytes 10 min after disruption of the organ structure of the spleens and is down-regulated after 2 h and later. In nuclear run-on experiments with nonstimulated lymphocytes there is already significant transcription of the first exon of c-myc, but almost no elongation of the transcript to exon 2 and 3. In concanavalin A-treated lymphocytes elongation is stimulated about 5-fold within 6 h and returns to background levels at 48 h post Stimulation. · The nuclear run-on analyses of nonactivated lymphocytes showed a signal for RNA complementary to c-myc mRNA detected with a probe specific for the exon 1/intron 1 boundary of c-myc, which disappeared with increasing time of concanavalin A Stimulation. This anti-sense transcription may play a role in regulating the elongation of cmyc transcripts.
Recombinant clonesthat represent the 3' part ofthe genome of the human spumaretrovirus (foamy virus) were established from viral DNA and from DNA complementary to viral RNA. The recombinant clones were characterized by blot hybridizations and nucleotide sequence analysis. The deduced protein sequence of the clones at their 5' ends was found to be homologous to the 3' domain of retroviral reverse transcriptases. Downstream of a small intergerne pol-env region a long open reading frame of 985 amino acid residues was identified that according to its genomic location, size, glycosylation signals, and hydrophobicity protile closely resembles the lentiviral env genes. The spumaretroviral env gene is followed by two open reading frames, termed bel-l and bel-2 which are located between env and the long terminal repeat region. The long terminal repeat of 1259 nucleotides is preceded by a polypurine tract and contains the canonical signal sequences characteristic for transcriptional regulation of retroviruses. The provisional classitication of the spumaretrovirus subfamily is discussed.
DNA ofhuman spumaretrovirus (HSRV) was cloned from both cDNA and from viral DNA into phage A and bacterial plasmid vectors. The recombinant plasm.ids harboring viral DNA were characterized by Southern blot hybridization and restriction mapping. Physical maps were constructed from cDNA and found to be colinear with the restriction maps obtained from viral DNA. The recombinant clones isolated contained viral DNA inserts which rangein size from 2.2 kb to 15.4 kb. The recombinant clones allowed to construct a physical map of the complete HSRV provirus of 12.2 kb.
During molecular cloning of proviral DNA of human. spumaretroVirus, various recombinant clones were estabUshed and analyzed. Blot hybridization revealed that one of the recoinbinant plasmids bad the characteristic features of a member of the long interspersed repetitive sequences famlly. The DNA element was analyzed by restrictioil mapping and nuelootide sequencing. It showed a high degree of amino acid sequence homology of 54.3% when conipared with the 5'-terminal part of the pol gelie product of the murine retrotransposon LIMd. The 3' region of the cloned DNA element encodes proteins witb an even higher degree of homology of 67.4% in comparison to the corresponding parts of a member of the primate Kpnl sequence family.
By means of indirect immunofluorescence analysis we investigated the effect of HIV -1 infection on HLA class I surface antigens. We report here that in CD4\(^+\) HeLa cells, in H9 cells, and in peripheral T Iymphocytes HLA class I antigens are down regulated following infection with HIV -1. The downregulation is effected at a posttranscriptional level since the amounts of HLA class I specific mRNA are similar in infected and uninfected cells. This phenomenon is not only correlated with the state of infection, that is, the presence of P24 of HIV-l in the cells, but also depends on the time of infection. Upon HLA class I downregulation by HIV infection, the specific lysis of peripheral blood cells by allogeneic CTL is reduced.
To probe into the functional properties of the major peripheral myelin cell surface glycoprotein P 0 , its ability to confer adhesion and neurite outgrowth-promoting properfies was studied in cell culture. Tothis aim, Po was expressed as integral membrane glycoprotein at the surface of CV -1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin-like extracellular domain of P0 (P0 -ED) was expressed as soluble profein in a bacterial expression system and used as substrafe coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth-promoting assays. The adhesion of P0 -expressing CV-1 cells to P0 -ED substrafe was specifically inhibitable by polyclonal Po antibodies (54% :t 6% ). In addition, the specific interaction between Po molecules could be reduced ( 49% ± 8%) by adding soluble P0 -ED to the culture medium, demonstrating that the homophilic inter~ction between recombinant Po molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig-like domain. Substrate-coated p -ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 ,_..m. This neurite outgrowth was specifically inhibitable by soluble P" (74% ± 14%) and P 0 antibodies (65% ± 9% ). These observations indicate that Po is capable of displaying two different types of functional roles in the myelination process of . peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interacton may indicate its roJe in the selfrecognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.
DNAs from peripheral blood mononuclear cells (PBMCs) of 21 patients with multiple sclerosis (MS), 1 patient with tropical spastic paraparesis (TSP) as well as DNAs from brain and spinal cord of 5 MS cases and 3 controls were examined for human T-cell lymphotropic virus (HTLV)-related sequences by polymerase chain reaction. The primers used were derived from the HTLV-1 gag, env and tax genes. Amplified products were separated on agarase gels, blotted onto nylon membranes and hybridized to specific radiolabelled oligonucleotides. The sensitivity of amplification and hybridization was one copy of target DNA in 10\8^5\) cellular genomes. None of the specimens was positive for HTLV-1 sequences except the TSP probe. These negative data are all the more significant because brain -material from MS patients was used in these studies. Our studies thus fail to support speculations that HTLV-I is involved in the aetiology of multiple sclerosis.
We have identified the major immunogenic structural proteins of the human foamy virus (HFV), a distinct member of the foamy virus subfamily of Retroviridae. Radiolabelied viral proteins were immunoprecipitated from HFV -infected cells by foamy virus antisera of human and non-human primate origin. Precipitated viral proteins were in the range of 31 K to 170K. Labelling of proteins with [\(^{14}\)C]glucosamine or with [\(^{35}\)S]methionine in the presence oftunicamycin, as well as endo-ß-N-acetylglycosaminidase Hand F treatment of [\(^{35}\)S]methionine-labelled proteins, revealed three viral glycoproteins of approximately 170K, 130K and 47K, most likely representing the env gene-encoded precursor, the surface glycoprotein and the transmembrane protein of HFV, respectively.
The long terminal repeat (LTR) of the human spumaretrovirus (HSRV) was examined with respect to its ability to function as transcriptional promotor in virus-infected and uninfected cells. Transient transfections using a plasmid in which the 3' L TR of HSRV was coupled to the bacterial chloramphenicol cetyltransferase (cat) gene revealed that the Ievei of HSRV LTR-directed cat gene expression was markedly increased in HSRV-infected cells compared to uninfected cells. Northern blot analysis of cat mRNA from transfected cultures suggests that transactivation of HSRVdirected gene expression occurs at the transcriptionallevel.
An infectious molecular clone (pHSRV) of the human Spumaretrovirus (HSRV) was constructed using viral DNA and cDNA clones. The infectivity of pHSRV was proven by transfection of cell cultures and subsequent infection of susceptible cultures with cell free transfection derlved virus. pHSRV derived virus produced foamy virus typical cytopathic effects in susceptible cultures. lnfected cells could be stained specifically with foamy virus antisera by means of indirect immunofluorescence. Radiolmmunoprecipltatlon revealed the presence of characteristic HSRV structural proteins in pHSRV infected cultures. By cotransfection of pHSRV and an indicator plasmid it was found that pHSRV is able to transactivate the viral L TR. Viral transcripts were found to be approximately 200 bases Ionger in pHSRV infected cultures compared to wildtype infected cultures. This difference is most likely due to an Insertion of DNA of non-viral origin ln the U3 region of the 3'L TR of the infectious clone.
The human foamy virus (HFV) genome possesses three open reading frames (bel I, 2, and 3) located between env and the 3' long terminal repeat. By analogy to other human retroviruses this region was selected as the most Iikely candidate to encode the viral transactivator. ResuIts presented here confirmed this and showed further that a deletion introduced only into the bell open reading frame of a plasmid derived from an infectious molecular clone of HFV abolished transactivation. In contrast, deletions in bel 2 and bel 3 had only minor effects on the ability to transactivate. The role of the bel I genomic region as a transactivator was further investigated by eukaryotic expression of a genome fragment of HFV spanning the bel I open reading frame. A construct expressing bell under control of a heterologous promoter was found to transactivate the HFV long terminal repeat in a dose-dependent fashion. Furthermore, it is shown that the U3 region of the HFV long terminal repeat is sufficient to respond to the HFV transactivator.
To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-y (lFN-y) and tumor necrosis factor-a (TNF-a) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-y and TNF-a synergistically inhibited the neurite outgrowth-promoting properties of the Schwann cells by spedfically dowllregulating myelin-associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAG inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by wh ich inflammatory cytokines interfere with Schwann cell-neuron interactions.
Transcription factor AP-1 modulates the activity of the human foamy virus long terminal repeat
(1991)
The human foamy virus (HFV) contains within the UJ region of its long terminal repeat (L TR) three perfect consensus sequences for the binding of the inducible transcription factor AP-1. Results of DNase I footprint protection and gel retardation assays demonstrated that proteins in extracts of HeLa and BHK-21 cells as weil as bacterially expressed Jun and Fos proteins bind to these AP-1 sites. By conducting transient expression assays using chloramphenicol acetyltransferase plasmids carrying LTR sequences with point-mutated AP-1 sites it was found that the three AP-1 sites contribute to the optimal activity ofthe HFV promoter. It is shown that lnduction of the HFV L TR by 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum factors is mediated through the AP-1 sites.
Progressive encephalopathy and myopathy in transgenic mice expressing human foamy virus genes
(1991)
Transgenie mice carrying the bel region of human foamy retrovirus (HFV) under transcriptional control of its own long terminal repeat expressed tbe transgene in their centrat nervous systems and in smootb and striated muscle tissues. The animals developed a progressive degenerative disease of tbe centrat nervous system and of the striated muscle. Because expression of tbe transgene was dosely correlated witb the appearance of structural damage and inflammatory reactions were scanty, the disease is likely to be caused directly by tbe HFV proteins. These unexpected findings call for a reevaluation of tbe patbogenic potential of HFV in humans.
Human immunodeficiency virus (HIV-1) infection in the human brain Ieads to characteristic neuropathological changes, which may result indirectly from interactions of the envelope glycoprotein gp 120 with neurons and/or glial cells. We therefore investigated the binding of recombinant gp120 (rgp120) to human neural cells and its effect on int~acellular.s.ignallin~. Herewe pre~ent evidence that rgp120, besides binding to galactocerebroside or galactosyl-sulfatlde, spec1f1cally bmds to a protem receptor of a relative molecular mass of approximately 180,000 Da (180 kDa) pre~ent. on the CD4-negative glioma cells D-54, but not on Molt4 T lymphocytes. Binding of rgp120 to this receptor rap1dly 1nduced a tyrosine-specific protein kinase activity leading to tyrosine phosphorylation of 130- and 115-kDa p~oteins. The c~ncentration of intracellular calciumwas not affected by rgp120 in these cells. Our data suggest a novel Signal transduc1ng HIV-1 gp120 receptor on CD4-negative glial cells, which may contribute to the neuropathological changes observed in HIV-1-infected brains.
HIV infection of CD4+ peripheral blood lymphocytes leads to a loss of MHC dass I molecules on the surface of the infected cells as detectable by monodonal antibody staining and flow cytometry. Incubation of the infected cells at 26 oe or treatment at 37 oe with peptides leads to upregulation of MHC dass I to levels equal to those found on uninfected cells cultured und er the same conditions. The data suggest that, after HIV infection, the mechanisms responsible for peptide generation, peptide transport and thus stable association between peptides and MHC dass I molecules are severely affected.
Expression of human foamy virus is differentially regulated during development in transgenic mice
(1992)
Tbe human foamy virus (HFV) is a recently characterized member ofthe spumavirus family. Although no diseases have been unequivocally associated with HFV infection, expression of HFV regulatory genes in transgenie mice induces a characteristic aeute neuro degenerative disease and a myopathy. To better eharaeterize the sequenee of events leading to disease, and to gain a better understanding of the underlying pathogenetic meehanisms, we have analyzed in detail the transgene expression pattern during development. Transcription of a construet containing all regulatory elements and aneillary genes of mv was analyzed by in situ hybridization and was shown to occur in two distinct phases. At midgestation, low but widespread expression was first deteeted in eells of extraembryonie tissues. Later, various tissues originating from embryonie mesoderm, neuroeetoderm, and neural erest transeribed the transgene at moderate levels. However, expression deereased dramatically during late gestation and was suppressed shortly after birth. After a latency period of up to 5 weeks, transeription of the transgene resumed in single eelJs distributed irregularly in the central nervous system and in the skeletal museIe. By the age of 8 weeks, an increasing number of eells displayed much higher expression levels than in embryonie Iife and eventually underwent severe degenerative ehanges. These findings demonstrate that HFV transgene expression is differentially regulated in development and that HFV cytotoxicity may be dose-dependent. Such biphasic pattern of expression differs from that of murine retroviruses and may be explained by the specificity of HFV regulatory elements in combination with cellular faetors. Future studies of this model system should, therefore, provide novel insights in the mechanisms controlling retrovirallatency.
Aim: To examine peripheral blood and skeletal muscle from patients with chronic fadgue syndrome for exogenous retrovirus. Methods: Blood samples from 30 patients and muscle biopsy specimens of 15 patients were examined for retroviral sequences by DNA extraction, polymerase chain reacdon (PCR), and Southern blotting hybridisation. Sera were examined for human foamy virus by western immunoblotting and indirect immunofluorescence techniques. Results: No difference between the padent and control populations was found for any of the PCR primer sets used (gag, pol, env, and tax regions of HTLV VII). An endogenous gag band was observed in both the padent and control groups. All sera were negative for antibody to human foamy virus. Conclusion: The results indicate that there is no evidence of retroviral involvement in the chronic fatigue syndrome.
The expression of measles virus (MV) in six different permanent human glioma cell lines (D-54, U-251, U-138, U-105, U-373, and D-32) was analyzed. Although all celllines were permissive for productive replication of all MV strains tested, U-251, D-54, and D-32 cells spontaneously revealed restrictions of MV transcription similar to those observed for primary rat astroglial cells and brain tissue. In vitro differentiation of D-54 and U-251 cells by substances affecting tbe intracellular cyclic AMP Ievel caused a significant reduction of tbe expression of tbe viral proteins after 18, 72, and 144 b of infection. This pronounced restriction was not paralleled to a comparable Ievel by an inhibition of tbe syntbesis and biological activity in vitro of virus·specific mRNAs as sbown by quantitative Northem (RNA) blot analyses and in vitro translation. The block in viral protein syntbesis could not be attributed to tbe induction of type I interferon by any of tbe substances tested. Our findings indicate tbat down-regulation of MV gene expression in human brain cells can occur by a cell type-rlependent regulation of tbe viral mRNA transcription and a differentiation-dependent regulation of translation, botb of wbicb may be crucial for the establisbment of persistent MV infections in tbe centrat nervous system.
Quantification of the peripheral nerve myelin glycoprotein PO and antibodies to PO is difficult due to insolubility of PO in physiological solutions. We have overcome this problern by using the water-soluble recombinant form of the extracellular domain of PO (PO-ED) and describe newly developed assays which allow detection and quantitation of PO and antibodies to PO, in serum and cerebraspinal fluid (CSF). These sensitive and specific assays based on the ELISA technique were used to study humoral immune responses to PO during experimental autoimmune ("allergic") neuritis (EAN). In order to establish these tests, monoclonal antiborlies to different epitopes of rodent and human PO-ED were produced. A two-antibody sandwich-ELISA allowing quantitation of PO Oower detection Iimit of 0.5 ngjml or 30 fmoljml) and an antibody-capture ELISA (lower detection Iimit 1 ng specific antibody jml) to detect antiborlies to PO in serum and CSF were developed. EAN was induced in rats by active immunization with bovine myelin or the neuritogenic protein P2 or by adoptive transfer using P2 specific CD4 positive T cells. Serum and CSF were assayed for the presence of PO-ED and antibodies to PO-ED or P2. Antibodies to PO-ED were detected during active myelin-induced EAN, but not during P2-induced or adaptive transfer EAN. The anti-PO-ED antibodies in the CSF showed a correJation with disease activity. In contrast, in the same model antibodies to P2 persisted long after the disease ceased. No soluble PO-Iike fragments could be found in serum or CSF during any of the three types of EAN. We conclude that PO may be a B-eeil epitope in EAN. These findings warrant a screen for antibodies to PO-ED in human immune neuropathies.
The effect of measles virus (MV) infection on mRNA expression and protein synthesis of cytokines in human malignant glioma celllines (0-54 and U-251) was investigated. Primary MV infections led in both celllines to the induction of interleukin-1 fJ (ll-1 (3), interleukin-6 (IL-6), interferon-(3 (IFN-fJ), and tumor necrosis factor-a (TNF-a). ln contrast, persistently infected astrocytoma lines continually produced IL-6 (two out of 12 lines high Ievels) and IFN-ß, whereas only 1 out of 121ines synthesized TNF-a and none IL-1ß. The pathways for induction of IL-1fJ and TNF-a expression were not suppressed by the persistent MV infection, since IL-1ß and TNF-a could be induced by external stimuli Jike diacylglycerol analog plus calcium ionophore. lnterestingly, persistently infected astrocytoma cells synthesized considerably higher Ievels of ll-1ß and TNF-a than uninfected cells afteradditional external induction. These results suggest that in the centrat nervous system (CNS) of SSPE patients a percentage of persistently infected astrocytes may continually synthesize IL-6 and IFN-ß, and in the presence of additional external stimuli, as possibly provided by activated lymphocytes, might ovarexpress the inflammatory cytokines IL-1 ß and TNF-a. This may be of pathogenetic significance in CNS diseases associated with persistent MV infections.
Humanfoamy virus (HFV) is a retrovirus encoding structural genes and, like human immunodeficiency virus and human T ceU leukemia virus I, several anciUary reading frames collectively termed the belgenes. We have previously shown that HFV transgenic mice develop an encephalopathy with neuronal loss in hippocampus and cerebral cortex. We have now raised and characterized rabbit antisera to various recombinant portions of gag, pot, env, and bel-I, the viraltransactivator. Immunoreactivity for gag and bel-I was observed in nuclei and processes of hippocampal and cortical neurons before the onset of morphological lesions and correlated with the appearance of HFV mRNA. Astrocyte-derived multinucleated giant ceUs containing HFV proteins were present in the brain oftransgenic mice coexpressingfuU- length HFV genes but not in mice expressing truncated gag and env, suggesting that these genes contain afusogenic domain. Expression of fuU-length structural genes decreased the life expectancy oftransgenic mice, implying an a4Juvant rolefor these proteins in HFV-induced brain damage. (Am] Pathol 1993, 142:1061-1072)
Seven monoclonal antibodies were raised against the immunoglobulin-like extracellular domain of PO (POED), the major protein of peripheral nervous system myelin. Mice were immunized with purified recombinant rat PO-ED. After fusion, 7 clones (POI-P07) recognizing either recombinant, rat, mouse, or human PO-ED were selected by ELlS A and were characterized by Western blot, immunohistochemistry, and a competition assay. Antibodies belonged to the IgG or IgM class, and P04-P07, reacted with PO in fresh-frozen and paraffin-embedded sections of human or rat peripheral nerve, but not with myelin proteins of the central nervous system of either species. Epitope specificity of the antibodies was determined by a competition enzyme-linked immunosorbent assay (ELISA) and a direct ELlS A using short synthetic peptides spanning the entire extracellular domain of PO. These assays showed that POl and P02 exhibiting the same reaction pattern in Western blot and immunohistochemistry reacted with different distant epitopes of PO. Furthermore, the monoclonal antibodies P05 and P06 recognized 2 different epitopes in close proximity within the neuritogenic extracellular sequence of PO. This panel of monoclonal antibodies, each binding to a different epitope of the extracellular domain of PO, will be useful for in vitro and in vivo studies designed to explore the role of PO during myelination and in demyelinating diseases of the peripheral nervous system.
The human foamy virus (HFV) bel-l transactivator protein was expressed in insect cells by a recombinant baculovirus. For the generation of the recombinant baculovirus, Acbel-1, the bel-l gene of an HFV mutant was used, that bears truncations in the bel-l overlapping bel-2 open reading frame. Acbel-1 infected Sf9 cells produced high amounts of recombinant protein of the same electrophoretic mobility (36 kD) as bel-l expressed in mammalian cells. The baculovirus expressed bel-l proteinwas readily identified by a polyclonal rabbit serum directed against bel-1 in immunoblot assay. As in mammalian cells, bel-l was predominantly localized to the nucleus of Acbel-1 infected insect cells. The baculovirus expressed bel-1 proteinwill be of use to determine the action of this novel viral transactivator more precisely.
In vitro and in vivo infection of rhesus monkey microglial cells by simian immunodeficiency virus
(1993)
The observation that microglial cells in brain tissue are probably a major target for human immunodeficiency virus (HIV) infection has raised interest in the pathogenic role of this cell population for the development of neuro-AIOS. Since it is very difficult to obtain microglia from normal or diseased human brain we studied microglial cells isolated from fresh brain tissue of uninfected and simian immunodeficiency virus (SIV) infected rhesus monkeys (Macacca mulatta) in comparison to peripheral blood macrophages. Besides the characterization of the phenotypes of these two cell populations, we examined the replication of SIV in the cells in addition to the effect of viral infection on the expression of cell surface molecules. We found that microglia and macrophages support replication of the wild-type SIV\(_{mac25}\), strain as well as the infectious clone (SIV\(_239\)). Infectious viruswas produced and a CPE developed. Isolated microglial cells from SIV-infected monkeys were latently infected independent of the presence of neuropathological lesions and produced infectious virus after 20-25 days in culture. In situ hybridization revealed that only a small percentage of isolated microglial cells are productively infected in vivo, yet the majority of these expressed MHC class II molecules. This indicated a state of activation that is acquired in vivo. These findings indicate that microglia are a prime target cell for SIV infection in CNS tissue.
Human foamy viruspol gene fragments were molecularly cloned into a procaryotic expression vector. The expression pattern of the cloned fragments and nucleotide sequence analysis of the 5' pol gene region revealed that in HFV the protease (PR) is located in the pol open reading frame. Purified recombinant proteins were used to generate antibodies in rats. ln immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (M,) of 127K was identified by antibodies directed against the reverse transcriptase (RT), RNaseH, or integrase (IN) domeins of pol. With concentrated virus as antigen, the RT and RNaseH antibodies recognized a protein of 80K, the IN antiserum recognized a protein of 40K, and the PR antiserum detected a protein of approximately 10K.
The role of the thymus in the pathogenesis of simian acquired immunodeficiency syndrome was investigated in 18 juvenile rhesus monkeys (Macaca mulatta). The thymus was infected from the first week post-SIVmac inoculation, but the amount of virus-positive cells was very low « 1 in 1 04 T cells) as demonstrated by polymerase chain reaction and in situ hybridization. First morphological alteration was a narrowing of the cortex at 12 and 24 wpi. Morphometry revealed no increase of pyknotic T cells but a decrease of the proliferation rate andflow cytometry showed a reduction of the immature \(CD4^+/CD8^+\) double-positive T cells. Ultrastructural analysis revealed vacuolization, shrinkage, andfinally cytolysis of the cortical epithelial cells and the interdigitating dendritic cells. Immunofluorescence staining exhibited a widespread loss of cortical epithelial cells. This damage to the thymic microenvironment could explain the breakdown of the intrathymic T cell proliferation. It preceded fully developed simian acquired immunodeficiency syndrome and is therefore considered to play a major role in its pathogenesis.
Transforming growth factor \(\beta\) (TGF-\(\beta\)) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-\(\beta\) on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (protoenhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-\(\beta\)-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-\(\beta\) and cyclosporin A in E14 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.
Phenotypic and functional changes in lymphocytes from rhesus monkeys (Macaca mulatta) were investigated during the first 6 months after infection with SIVmac 32H. Animals preimmunized with keyhole limpet hemocyanin (KLH) were sacrificed l, 3, 6, 12, and 24 weeks post infection. Subset composition and function of lymphocytes from blood, spleen, lymph node and thymus were analysed. In addition to a rapid decline in CD4/CD8 ratios, a massive reduction in CD29+CD4+ cells was seen in the periphery. Although depletion of this subset was observed throughout the course of this experiment, the loss of proliferative T cell responses was most pronounced very early after infection and partially recovered after Month 3. Polyclonal cytotoxic responses were only slightly affected. In the thymus, a gradual, but moderate loss of CD4 + CD8 + immature thymocytes, and a relative increase in both CD4 + and CD8 + mature subsets was observed. Infectious virus was readily recovered from homogenates of lymph node and spleen, but not of thymus tissue. Interestingly, however, virus was detected in thymocytes from all infected animals by cocultivation with a simian immunodeficiency virus (SIV) susceptible cell line.
The thymus in SIV infection
(1993)
no abstract available