Ubiquitylated Tax targets and binds the IKK signalosome at the centrosome.

Constitutive activation of the NF-kappaB pathway by the Tax oncoprotein plays a crucial role in the proliferation and transformation of HTLV-I infected T lymphocytes. We have previously shown that Tax ubiquitylation on C-terminal lysines is critical for binding of Tax to IkappaB kinase (IKK) and its subsequent activation. Here, we report that ubiquitylated Tax is not associated with active cytosolic IKK subunits, but binds endogenous IKK-alpha, -beta, -gamma, targeting them to the centrosome. K63-ubiquitylated Tax colocalizes at the centrosome with IKK-gamma, while K48-ubiquitylated Tax is stabilized upon proteasome inhibition. Altogether, these results support a model in which K63-ubiquitylated Tax activates IKK in a centrosome-associated signalosome, leading to the production of Tax-free active cytoplasmic IKK. These observations highlight an unsuspected link between Tax-induced IKK activation and the centrosome.

[Foamy virus, an original retroviral model]. / Les virus foamy, un modèle rétroviral original.

Foamy viruses (FVs) or spumaviruses are complex retroviruses isolated in several mammal species like cats, cattle and horses. Highly prevalent in non-human primates they are not naturally present in humans, although several cases of simian-to-human transmissions have been described. Interestingly, the replication strategy of FVs differs in many aspects from that of other retroviruses, presenting features that are closely related to pararetroviruses, exemplified by the hepatitis B virus (HBV), but also characteristics that are closely related to yeast retrotransposons leding to the creation of the distinct Spumaretrovirina subfamily.

Human foamy virus capsid formation requires an interaction domain in the N terminus of Gag.

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly.

Isolation and characterization of an equine foamy virus.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.

An evolutionarily conserved splice generates a secreted env-Bet fusion protein during human foamy virus infection.

Foamy viruses (spumaretroviruses) represent a retroviral genus which exhibits unusual features relating it to pararetroviruses. Previously, we reported the existence of a protein species harboring Env, Bel, and Bet epitopes in human foamy virus (HFV)-infected cells (M. L. Giron, F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Périès, and R. Emanoil-Ravier, J. Virol. 67:3596-3600, 1993). Here, we identify this protein as a 160-kDa Env-Bet fusion glycoprotein (gp160) translated from an mRNA species harboring a highly conserved splice site which deletes the membrane anchor domain of Env and fuses the env open reading frame with that of bel1/bet. While gp160 and Bet proteins were both secreted into the supernatant, only Bet was taken up by recipient cells. Since Bet plays a key role in the switch from lytic to chronic infection, secretion of Bet and gp160, together with cellular uptake of Bet, could be highly relevant for both immune response and development of HFV infection in vivo.

Long-term persistent infection of domestic rabbits by the human foamy virus.

Human foamy virus (HFV) belongs to the spumaretrovirus group of the Retroviridae taxonomic family. Attempts to associate HFV or other foamy viruses to a specific pathology still remain unsuccessful. However, viral gene expression as well as tissue-specific tropism in an in vivo context remain poorly analyzed. To address this issue, we have infected domestic rabbits with a single dose of HFV and followed them at the biological and molecular levels for 5 years. No apparent pathology was detectable in the infected animals which have developed a strong immunological response against major viral proteins. We found that HFV provirus in blood cells and several organs persisted predominantly in its defective form, delta HFV, suggesting that in vivo viral persistence could be related to homologous interference as was recently shown in vitro. This animal model might be useful for studying the in vivo targets of HFV and should also be convenient for testing therapeutic effects of antiretroviral drugs.

Expression and maturation of human foamy virus Gag precursor polypeptides.

In this report, we address the processing of the Gag polypeptides of human foamy virus previously reported to be atypical. In the cytoplasm or the nucleus of infected cells as well as in free virus particles, two Gag precursor polypeptides were identified at approximately 72 and 68 kDa, p72 giving rise to p68 by a maturation process. Efficient maturation of Gag precursors was observed only in two situations: (i) during the early steps of virus adsorption and (ii) under experimental conditions, including treatment with DNase I, known to dissociate actin polymers associated with high ionic strength and ionic detergents. Rather than being a defective viral protease function, an association of Gag precursors with a cytoskeleton network might be responsible for the low rate of Gag protein maturation through inhibition of their cleavage by the protease.

In vitro studies on interferon-inducing capacity and sensitivity to IFN of human foamy virus.

We demonstrate in this article that human foamy virus (HFV) fails to induce interferon (IFN) production in two different human tissue culture cell lines: U373-MG and AV3. We also show the effect of human alpha-, beta- and gamma IFN on the multiplication cycle of HFV. Treatment of cells with 100 IU/ml of any IFN led to strong inhibition of an HFV-induced cytopathic effect. This effect was associated with a significant diminution of reverse transcriptase activity in supernatant fluids of IFN-treated infected cultures, and a substantial decrease in viral particle production, as detected by electron microscopy. All these effects were accompanied by strong inhibition of both viral proteins and RNA synthesis, as well as almost total disappearance of free and integrated proviral DNA. In light of our data, human IFN action on HFV seems to be mediated by a mechanism which differs from that observed in the case of other retroviruses (type C and D for instance); however, it evokes that described for HIV.

Human foamy virus DNA forms and expression in persistently infected Dami megakaryocytic cells.

We have characterized human foamy virus (HFV) proviral DNA and determined HFV expression in a persistent infection model, the Dami megakaryocytic cell line. Molecular studies were performed on parental persistently infected cells (Dami-P), as well as on derived clones (Dami-Cl). We report that in these nonlytic and non-HFV producer cells, viral DNA was found to be integrated into the cellular genome and that the few free proviral forms detected in Dami-P cells were deleted in their 5' LTR. Our molecular analysis indicates the presence of undeleted 5' LTR forms in the integrated provirus within a proviral population mainly composed of deleted forms. In addition, the deletion in the bel1 trans-activator gene, previously described by Saïb et al., was found to be highly predominant. However, in 5-iodo-2'-deoxyuridine treated Dami-Cl cultures, virus production occurred, providing evidence for the presence of complete viral genome. Analysis of HFV expression in Dami-Cl cells, by Northern blot and immunoprecipitation, shows that the most striking difference between cytolytic and persistent HFV infection was the lack of expression of structural viral proteins, in contrast with Bet protein expression, which is maintained. Our data suggest that the Bet protein could be involved in the maintenance of viral persistency and that the persistently infected Dami system provides a suitable model for clarifying its function.

Comparative studies on IL-6 production status in HTLV-1 chronically infected cell lines derived from different HTLV-1 associated pathologies.

We have comparatively studied the qualitative and quantitative status of Interleukin 6 (IL-6) production in several HTLV-1 chronically infected T-cell lines derived from patients suffering either of adult-T-cell leukemia/lymphoma or of tropical spastic paraparesis (TSP). Two other HTLV-1 chronically infected cell lines, coming from healthy seropositive carriers, were also analyzed. Our results demonstrate that, independently of the origin, all these T cell lines, as compared to uninfected HTLV-1 T cell ones, which were always IL-6 non producers, synthesize and yield comparable amounts of IL-6 synthesis, he nosological origin of infecting viral strains does not seem to play a differential role on IL-6 qualitative or quantitative production parameters.

Human foamy virus polypeptides: identification of env and bel gene products.

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product.

Molecular differences between two immunologically related spumaretroviruses: the human prototype HSRV and the chimpanzee isolate SFV6.

A comparative study at the genomic and protein level was performed between two immunologically related spumaretroviruses, the human HSRV and the simian SFV6. Cross immunoprecipitation analysis with specific polyclonal and monoclonal antisera indicates shared antigenic determinants. However, restriction analysis of the viral DNAs and thermal stability of the hybrids demonstrate that HSRV and SFV6 are two different isolates.

Cell surface phenotype and human T lymphotropic virus type 1 antigen expression in 12 T cell lines derived from peripheral blood and cerebrospinal fluid of West Indian, Guyanese and African patients with tropical spastic paraparesis.

Twelve long-term cell lines were established from peripheral blood mononuclear cells (PBMC) or cerebrospinal fluid cells of patients with human T lymphotropic virus type I (HTLV-1) seropositive tropical spastic paraparesis (TSP) originating from the French West Indies, French Guyana or the Central African Republic. Most of these long-term interleukin-2-dependent cell lines exhibited a pattern characteristic of CD4(+)-activated T cells with high expression of CD2, CD3 and CD4 antigens, associated with a strong density of TAC and DR molecules. Nevertheless, in five cases CD8 expression was present at a significant level. HTLV-I antigens were never detected in uncultured PBMC, but they were expressed in a few cells after short-term culture and after 4 months the majority of the cells were HTLV-I positive, as demonstrated by indirect immunofluorescence (IF) using polyclonal or monoclonal anti-p19 and anti-p24 antibodies. Low and variable levels of reverse transcriptase activity were detected in supernatant fluids of these cell lines only after 4 months of culture, when at least 50% of the cells exhibited HTLV-I antigens by IF. However, numerous type C HTLV-I-like viral particles were detected, mostly in the extracellular spaces, with rare budding particles. Similar findings were found in three T cell lines derived from West Indian and African patients with adult T-cell leukaemia/lymphoma (ATLL). Differences in high Mr polypeptides were detected by Western blot in cell lysates when comparing TSP- or ATLL-derived T cell lines. Thus a signal of 62K was easily detectable in all the TSP lines, but not in the ATLL lines. In all cell lines bands corresponding to p53, p24 and p19 viral core polypeptides were present, as was the env gene-coded protein p46.

alpha-IFN treatment does not induce Ki-ras expression in hairy-cell leukemia patients.

alpha-Interferon (IFN) is effective in the treatment of hairy-cell leukemia (HCL), but the treatment is sometimes over a long period. Biological changes such as the increase of tumorigenicity can occur rapidly in vivo as a result of beginning this treatment; an increase in c-Ki-ras oncogene expression has also been observed. In order to determine whether the findings observed in vitro would be duplicated in an in vivo system, we decided to analyze the Ki-ras RNA and protein levels in the lymphocytes of three HCL patients, compared with these levels in seven normal donors and one non-treated HCL patients. Ki-ras was not activated by IFN, at least not in lymphocytes. Therefore, the data suggest that the drug could be used for long-term therapy with relatively low risk to the patients.

[Establishment of a T lymphoid cell line producing a retrovirus of HTLV-I type, from peripheral blood of a patient with tropical spastic paraparesis]. / Etablissement d'une lignée lymphoïde T productrice d'un rétrovirus de type HTLV-I à partir du sang périphérique d'un patient atteint de paraparésie spastique tropicale.

HTLV-I, the causative agent of Adult T cell Leukemia, has recently been found to be associated with chronic neuromyelopathies common in tropical areas and in Japan. We report here the establishment of a lymphoid T cell line from peripheral blood lymphocytes of a patient with Tropical Spastic Paraparesis. This cell line produces a retrovirus whose morphologic, antigenic and genetic characteristics show no detectable difference from the leukemogenic prototype of HTLV-I.

Isolation of high-molecular-weight DNA from eukaryotic cells by formamide treatment and dialysis.

A new method for the isolation of high-molecular-weight DNA from eukaryotic cells is presented. It is based on formamide treatment and extensive dialysis of cellular proteinase K digests. This procedure consists mainly of a series of incubations allowing simultaneous preparation of several DNAs which can then be restricted, ligated, hybridized, and cloned.

Polyadenylation of ribonucleic acids in mouse L cells infected by encephalomyocarditis virus.

Polyadenylation of cytoplasmic RNAs was investigated in L cells infected with encephalomyocarditis virus. During the early stage of infection, the mean size of newly made poly(A) chains attached to cell mRNAs decreased gradually, in parallel with the shut-off of RNA synthesis. Later on, poly(A) segments, an average of 50 nucleotides long, were associated with viral RNA isolated either from polysomes or whole cytoplasm. These segments seemed to be processed differently in the course of time, and RNA molecules containing short poly(A) tracts (less than 20 bases) were incorporated into the virions.

Size of the poly(A) sequences in encephalomyocarditis virus RNA.

Poly(A) sequences were demonstrated in the RNA of encephalomyocarditis (EMC) virus by affinity chromatography on poly(U)-Sepharose, treatment with RNases, and determination of the ability of the RNA to prime for reverse transcription. The size of the poly(A) sequences was estimated as 10-20 nucleotides in length by determination of the fraction resistant to pancreatic RNases plus T1 RNases, followed by analysis of electrophoretic mobility on polyacrylamide gels and of the AMP-to-adenosine ratio after alkaline hydrolysis. The poly(A) sequences are located at the 3' end of the RNA as shown by mild digestion by snake venom phosphodiesterase.