Feline foamy virus (FeFV) belongs to the group of spumaretroviruses that contain in addition togag, pol,andenvaccessory genes collectively calledbelgenes. Primate FVs have been shown to utilize ...internal promoters in addition to the 5′ LTR promoters. In contrast to other known retroviruses, the FVpolgenes are expressed via spliced transcripts. Northern blot analysis and reverse transcription-coupled polymerase chain reactions (RT-PCR) were used to amplify, clone, and characterize cDNAs generated from subgenomic viral transcripts. Sequencing of the splice site junctions of the different FeFV mRNAs showed that singly and multiply spliced subgenomic transcripts were expressed in virus-infected cells. The relative amount of the splicedpol-specific transcripts was quantitated and FeFVpolmRNA found to be expressed at about one-half of that of the genomic mRNA. The major FeFV internal start site of transcription was identified at RNA position 7925. Comparison of the FeFV transcriptional patterns to those of the human foamy virus revealed that the FeFVbel 1mRNA was expressedexclusivelyfrom the internal promoter in contrast to primate foamy viruses that use both the LTR and the internal promoter for Bel 1 expression. Unexpectedly, anenv-bel 2mRNA was identified in FeFV-infected cells. In addition, cDNAs from FeFV-infected cells were directly amplified by PCR without RT reactions and found to correspond to genomic and to a subset of different subgenomic FeFV mRNAs.
This paper summarizes the results of the genetic analysis of several parts of the genome of canine distemper virus (CDV) field isolates and vaccine viruses. The haemagglutinin (H) gene analysis ...showed that recent viruses did not differ significantly from vaccine strains. The analysis of the long untranslated region between the matrix (M) and fusion (F) gene revealed distinct genetic heterogeneity. The putative F protein start codon AUG
461 of vaccine strain Onderstepoort was found to be mutated in all wild-type isolates and in another vaccine strain. The proximal coding part of the F gene was well conserved. Phylogenetic analysis of this segment showed the presence of several cocirculating CDV genotypes.
Serum samples collected from patients with a wide variety of diseases from African and other countries were tested for antibodies to the human spumaretrovirus (HSRV). A spumaviral env-specific ELISA ...was employed as screening test. Out of 3020 human sera screened, 106 were found to be positive (3.2%). While the majority of patients' sera from Europe (1581) were negative, 26 were positive (1.6%). Sera from healthy adult blood donors (609), from patients with multiple sclerosis (48), Graves' disease (45), and chronic fatigue syndrome (41) were negative or showed a very low prevalence for spumaviral env antibodies. A higher percentage of seropositives (6.3%) were found among 1338 African patients from Tanzania, Kenya, and Gabon. Out of 1180 patients from Tanzania, 708 suffered from tumors, 75 from AIDS, and 128 had gynecological problems; 51 of the Tanzanian patients were HSRV seropositive (4.3%). A particularly high percentage of 16.6% seropositives were identified among nasopharyngeal carcinoma patients (NPC) from Kenya and Tanzania consistent with results reported 10 years ago. However, 20 nasopharyngeal carcinoma patients from Malaysia were HSRV-seronegative. In selected cases, sera from seropositive individuals were reacted with proteins from HSRV-infected cells in vitro. HSRV env- and gag-specific antibodies were specifically detected by these sera in Western blots. The results indicate spumavirus infections in human patients with various diseases at a relatively low prevalence worldwide; in African patients, however, the prevalence of spumavirus infections is markedly higher.
The human foamy virus (HFV) protease (PR) was cloned into a modified thioredoxin fusion vector that carried a His-tag in the centrally located surface loop of theE. coli trxAprotein, bacterially ...expressed as a soluble fusion protein, and subsequently purified by affinity chromatography. By using HFV Gag protein substrates, the purified recombinant HFV PR was enzymatically active whereas the corresponding active site PR mutant Asp/Ala was inactive. Incubation of synthetic peptides containing residues that flank the putative cleavage site with the recombinant HFV PR and subsequent matrix-assisted laser desorption ionization mass spectrometry of the cleavage products identified the proteolytic processing site of the HFV Gag precursor p74 and revealed that the peptide sequence RAVNTVTQ was cleaved between the Asn and Thr bond.
Appropriate expression of HTLV-1 genes requires transcriptional transactivation by Tax and post-transcriptional regulation by Rex, both mediated by LTR encoded RNA sequences. Using a combination of ...deletion mutagenesis, Rex-reporter CAT assays, fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy it was established that in the absence of Rex, CAT mRNAs harboring HTLV-1 LTR sequences were unable to leave the nucleus. Deletion of the known U5 encoded cis-acting repressing sequence (CRS) led to a partial release of nuclear retention. A novel regulatory element overlapping the 3' Rex responsive element (RxRE) region was shown to prevent export and expression of these transcripts. Deletion of both the 5' LTR encoded CRS and 3' LTR encoded downstream repressive sequence (3' CRS) led to constitutive mRNA nuclear export and gene expression, independently of Rex. The locations of the two regulatory elements indicate that while the 5' CRS selectively acts to hinder export of unspliced transcripts, the 3' CRS has the capacity to induce nuclear retention of all HTLV-1 transcripts, and therefore could potentially contribute to viral latency in infected cells.
The hallmarks of the spumaretrovirus or human foamy virus (HFV) are summarized and discussed with special focus on the potentials to use HFV as a new retroviral vector system. The special features of ...HFV are the expression of pol by splicing and start of translation at a defined initiation codon. The first Met of Pol is conserved in the six known foamy virus genomic sequences. Another remarkable characteristic of HFV is the presence of a Gly‐Arg‐rich sequence instead of the Cys‐Cys motif of the classical retroviral nuclecapsid proteins. The preferential budding of HFV into cytoplasmic vesicles and the potential to exploit it in the application of corresponding vector systems is discussed. In addition, recent reports of transducing marker genes into susceptible cells will be reviewed. Stem Cells 1997; 15(suppl 1): 141‐147
Recombinant plasmid clones were constructed harbouring the central domains of the outer membrane protein and the transmembrane protein of the env gene of human spumaretrovirus (HSRV). The ...corresponding fusion proteins were expressed in E. coli, purified and used subsequently to produce antibodies against the HSRV env proteins in rabbits. The authenticity of the bacterially produced domain of the HSRV env proteins was shown by radioimmunoprecipitation of the viral env glycoprotein from HSRV-infected human cells with rabbit antibodies raised against the recombinant antigens. The recombinant viral antigens were used to establish a sensitive and spumavirus-specific enzyme-linked immunosorbent assay (ELISA). This anti HSRV antibody ELISA makes it possible to screen human sera for the presence of spumavirus infections.
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