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A Rethwilm

Publications and source records attributed to A Rethwilm.

At least 55 records · Page 3Linked to original sources
PubMed1995

Infectious proviral clones of chimpanzee foamy virus (SFVcpz) generated by long PCR reveal close functional relatedness to human foamy virus.

Infectious proviral clones of simian foamy virus isolated from chimpanzee (SFVcpz) were generated by long PCR. Two overlapping fragments representing the complete provirus were amplified from genomic DNA of infected cells. Four 8.8-kbp amplimers extending from base 1 of the provirus into the env gene and five 4.45-kbp amplimers reaching from env to the end of the 3'-LTR were cloned into pCR II. Subsequently, the proviral fragments were combined in a chessboard manner to generate 20 plasmids containing full-length proviral DNA. Four plasmids produced infectious virus after transfection of susceptible cells. A distinct proviral form bearing a deletion in the transactivator gene joining both exons of a second regulatory gene present in wild-type foamy virus-infected cells started to emerge 48 hr after transfection of BHK cells with infectious SFVcpz DNA. This observation supports a novel hypothesis to explain establishment of foamy virus latency. The transactivator protein Taf of SFVcpz transcomplemented for the homologous protein Bel-1 of the unique human foamy virus isolate (HFV) and Bel-1 exhibited the reciprocal activity, suggesting that HFV could represent a variant of chimpanzee foamy virus.

Animals↗
PubMed1995

Replicating foamy virus-based vectors directing high level expression of foreign genes.

Replication-competent retroviral vectors (pFOV-1 to -3 and -7) were constructed on the basis of an infectious human foamy virus molecular clone which has deletions in the U3 region of the long terminal repeat and in the 3' region of the genome, previously identified to be nonessential for virus replication in vitro. The CAT and luciferase indicator genes were expressed as C-terminal fusion proteins to 215 amino acids of the viral Bet protein in the pFOV-1 vector. Introduction of the foot-and-mouth disease 2A protease sequence between the truncated bet coding sequence and the cloning site for the insertion of foreign genes in the pFOV-7 vector resulted in self-cleaving of the recombinant fusion protein. Alternatively, an internal ribosomal binding site was introduced, allowing expression of authentic foreign protein (pFOV-2 and -3 vectors). DNA fragments derived from the mouse hepatitis virus surface gene up to the length of 1.3 kb were inserted into pFOV-1. The vector constructs gave rise to viruses which were fully infectious in diploid human fibroblasts and recombinant viruses stably expressed high levels of foreign protein indicating that the pFOV vectors may be useful tools to study the effects of proteins of interest at least in tissue culture cells.

Amino Acid Sequence↗
PubMed1995

A comparative study of higher primate foamy viruses, including a new virus from a gorilla.

Few foamy (spuma) retroviruses have been investigated in molecular detail, despite their previous isolation from several mamalian species, including ten neutralization serotypes from various primates. Here, we have studied a new gorilla foamy virus (SFV-Gg) and investigated its functional and phylogenetic relationship to the human (HFV) and other primate foamy viruses, including that recently described in orangutans (SFV-11). Nucleotide sequencing of PCR products obtained from the R/U5 region of the LTR, gag, and pol genes revealed a close relationship between HFV and three chimpanzee isolates (SFV-6, SFV-7, and SFV-cpz). The SFV-Gg, SFV-11, rhesus macaque (SFV-1), and African green monkey (SFV-3) isolates were more divergent. To explore functional relationships, primate foamy virus transactivation of HFV LTR driven beta-galactosidase expression in a newly constructed cell line, BHLL, was investigated. HFV, SFV-6, and SFV-7 potently transactivated HFV LTR driven lacZ gene expression, SFV-Gg induced expression approximately 10-fold less efficiently, and SFV types 1, 2, 3, and 11 did not significantly transactivate the HFV LTR. It was, thus, possible to assay serum neutralizing activity in SFV-infected primates against HFV, SFV-6, and SFV-7 by reduction of beta-galactosidase activity following infection of the indicator cell line. Sera from infected chimpanzees and gorillas neutralized, to varying degrees, each of these three viruses, whereas orangutan sera did not. Our results, based on DNA sequences and functional assays, support the conclusion that HFV is closely related to foamy viruses of chimpanzee origin.

Animals↗
PubMed1995

Markers of foamy virus infections in monkeys, apes, and accidentally infected humans: appropriate testing fails to confirm suspected foamy virus prevalence in humans.

Foamy viruses (FVs) persist in healthy individuals of various mammalian species, including nonhuman primates. Laboratory markers of FV infection are (1) virus in throat epithelium or peripheral blood lymphocytes (PBLs), (2) proviral DNA sequences in PBLs and various solid organs, and (3) antibodies reactive to viral antigens on Western blots, in radioimmunoprecipitation tests, and in immunofluorescence assays. Using PCR and serological tests, we readily detected FV markers in naturally infected African green monkeys, rhesus monkeys, and chimpanzees, as well as in accidentally infected humans. Transmission of simian foamy viruses to humans (by bite or inadvertent laboratory infection) leads to viral markers, without affecting the recipient. Reports on FV-associated clinical disorders (e.g., thyroid or neurological) have remained controversial. In this study we failed to detect, by PCR, viral sequences in the samples from 223 patients, including 16 HIV-infected Africans, 46 Graves' disease patients, and 28 patients with the de Quervain's thyroiditis. Evaluation of 2688 sera from suspected high-risk areas (e.g., Central and East Africa, or high-risk groups such as HIV-infected individuals and patients with AIDS, thyroid, and neurological disorders) did not reveal FV-specific antibodies in a single case. Previously reported FV seroprevalence in various populations has never been verified by appropriate confirmatory tests. The strain of "human foamy virus" has remained a unique isolate. In conclusion, FVs are unlikely--at present--to circulate in human populations.

Africa↗
PubMed1994

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Animals↗
PubMed1994

Nuclear localization of foamy virus Gag precursor protein.

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus.

Amino Acid Sequence↗
PubMed1994

[Correlation of organ pathology and distribution of virus replicating cells, demonstrated with RNA in situ hybridization of SIVmac infection of Macaca mulatta].

22 juvenile rhesus macaques were infected i.v. with SIVmac and killed at defined timepoints after infection. Productively infected cells were detected by RNA in situ hybridization in the paraffin material. Their number was correlated with the pathology of lymph nodes, thymus, extranodal lymphatic parenchyma and other organs. In the first weeks all lymphatic tissues and compartments got infected, as well as the brain, the bone marrow and other organs. The high virus replication during this first phase disappeared with the onset of the seroconversion and remained low during all stages of atrophy of the lymphatic parenchyma. The atrophy of the lymphatic parenchyma and its microenvironment was not correlated with virus replication. This may implicate that a virostatic therapy might be more successful in the first weeks of infection.

Animals↗
PubMed1993

Identification of pol-related gene products of human foamy virus.

Human foamy virus pol 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. In immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (M(r)) of 127K was identified by antibodies directed against the reverse transcriptase (RT), RNaseH, or integrase (IN) domains 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.

Base Sequence↗
PubMed1993

In vitro and in vivo infection of rhesus monkey microglial cells by simian immunodeficiency virus.

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-AIDS. 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 SIVmac251 strain as well as the infectious clone (SIV239). Infectious virus was 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.

Animals↗
PubMed1993

BEL-1 transactivator responsive sequences in the long terminal repeat of human foamy virus.

Cis-regulatory elements in the long terminal repeat (LTR) of human foamy virus (HFV) were identified by using LTR mutants to transiently express the chloramphenicol acetyl-transferase gene after co-transfection with an expression plasmid for the virus bel-1 (transactivator) gene. The R-U5 region and an element in the 5' U3 region were found to negatively influence HFV gene expression. The complete BEL-1 responsive region was mapped to extend from nucleotide position -471 to position -93 relative to the start of transcription. Within this region, three elements were identified that in the homologous or a heterologous (SV40) promoter context can, independently and irrespective of their orientation, act as targets for BEL-1. These elements are located between nucleotide positions -413/-378, -361/-291, and -124/93. The target elements do not share obvious sequence homologies. The mechanism of HFV transactivation appears to be novel among the complex retroviruses and is likely to involve, as yet, undiscovered cellular DNA binding factors.

Animals↗
PubMed1993

Expression of the human foamy virus bel-1 transactivator in insect cells.

The human foamy virus (HFV) bel-1 transactivator protein was expressed in insect cells by a recombinant baculovirus. For the generation of the recombinant baculovirus, Acbel-1, the bel-1 gene of an HFV mutant was used, that bears truncations in the bel-1 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-1 expressed in mammalian cells. The baculovirus expressed bel-1 protein was readily identified by a polyclonal rabbit serum directed against bel-1 in immunoblot assay. As in mammalian cells, bel-1 was predominantly localized to the nucleus of Acbel-1 infected insect cells. The baculovirus expressed bel-1 protein will be of use to determine the action of this novel viral transactivator more precisely.

Animals↗
PubMed1993

Functional analysis of human foamy virus accessory reading frames.

Foamy viruses belong to the retroviruses which possess a complex genome structure. The human foamy virus (HFV) isolate bears three open reading frames (the so-called bel genes) in the 3' region of the genome which have been reported to give rise to possibly six different proteins via alternative splicing (W. Muranyi and R. M. Flügel, J. Virol. 65:727-735, 1991). In order to analyze the requirements of these proteins for HFV replication in vitro, we constructed a set of single and combinatory bel gene mutants of an infectious molecular clone of HFV. The mutant which lacked the transacting activator, bel-1, was found to be replication incompetent. All other mutants replicated equally well and gave rise to comparable titers of infectious cell-free virus. When HFV proviruses were put under the control of a heterologous promoter (simian virus 40), none of the accessory gene products was found to be required for expression of structural (gag) proteins. There was no evidence for a posttranscriptional regulatory protein that is present in other complex retroviruses.

Base Sequence↗
PubMed1993

Alterations of thymus cortical epithelium and interdigitating dendritic cells but no increase of thymocyte cell death in the early course of simian immunodeficiency virus infection.

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 10(4) 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 and flow cytometry showed a reduction of the immature CD4+/CD8+ double-positive T cells. Ultrastructural analysis revealed vacuolization, shrinkage, and finally 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.

Animals↗
PubMed1993

Human foamy virus proteins accumulate in neurons and induce multinucleated giant cells in the brain of transgenic mice.

Human foamy virus (HFV) is a retrovirus encoding structural genes and, like human immunodeficiency virus and human T cell leukemia virus I, several ancillary reading frames collectively termed the be1 genes. 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, pol, env, and bel-1, the viral trans-activator. Immunoreactivity for gag and bel-1 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 cells containing HFV proteins were present in the brain of transgenic mice coexpressing full-length HFV genes but not in mice expressing truncated gag and env, suggesting that these genes contain a fusogenic domain. Expression of full-length structural genes decreased the life expectancy of transgenic mice, implying an adjuvant role for these proteins in HFV-induced brain damage.

Animals↗
PubMed1992

Search for retrovirus in the chronic fatigue syndrome.

AIM: To examine peripheral blood and skeletal muscle from patients with chronic fatigue 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 reaction (PCR), and Southern blotting hybridisation. Sera were examined for human foamy virus by western immunoblotting and indirect immunofluorescence techniques. RESULTS: No differences between the patient and control populations was found for any of the PCR primer sets used (gag, pol, env, and tax regions of HTLV I/II). An endogenous gag band was observed in both the patient 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.

Blotting, Southern↗
PubMed1992

Expression of human foamy virus is differentially regulated during development in transgenic mice.

The human foamy virus (HFV) is a recently characterized member of the spumavirus family. Although no diseases have been unequivocally associated with HFV infection, expression of HFV regulatory genes in transgenic mice induces a characteristic acute neurodegenerative disease and a myopathy. To better characterize the sequence of events leading to disease, and to gain a better understanding of the underlying pathogenetic mechanisms, we have analyzed in detail the transgene expression pattern during development. Transcription of a construct containing all regulatory elements and ancillary genes of HFV was analyzed by in situ hybridization and was shown to occur in two distinct phases. At midgestation, low but widespread expression was first detected in cells of extraembryonic tissues. Later, various tissues originating from embryonic mesoderm, neuroectoderm, and neural crest transcribed the transgene at moderate levels. However, expression decreased dramatically during late gestation and was suppressed shortly after birth. After a latency period of up to 5 weeks, transcription of the transgene resumed in single cells distributed irregularly in the central nervous system and in the skeletal muscle. By the age of 8 weeks, an increasing number of cells displayed much higher expression levels than in embryonic life and eventually underwent severe degenerative changes. 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 factors. Future studies of this model system should, therefore, provide novel insights in the mechanisms controlling retroviral latency.

Animals↗