Proteolytic processing of foamy virus Gag and Pol proteins.

The foamy viral proteases (FV PRs) are set apart from other retroviral processing enzymes by unique features. The first remarkable property is that FV PRs are enzymatically active as high-molecular-mass Pro-Pol proteins. Hence there exist multiple forms of active FV PRs that likely contribute to cleavage site specificity. A FV PR of low molecular size is not detectable in purified virions, in contrast to PRs of other retroviruses that are found in virus particles. Because the major part of Pol remains attached to the amino-terminal enzymatically active PR protein region, the FV-specific way of expressing Pro-Pol polyproteins from a pol-specific transcript provides for the incorporation of Pro-Pol and IN into virus particles. Proteolytic processing of Gag and Pol proteins is incomplete and delayed. Another novel feature is that the catalytic center of the active dimers of cat FV PR consists of D-S/T-Q instead of D-S/T-G, an unprecedented feature of this enzyme. The temporal and spatial control and the factors that regulate FV PRs remain to be elucidated.

Activation of HTLV-I long terminal repeat by stress-inducing agents and protection of HTLV-I-infected T-cells from apoptosis by the viral tax protein.

HTLV-I is etiologically implicated with tropical spastic paraparesis/HTLV-I associated myelopathy, adult T-cell leukemia and certain other diseases. However, after infection the virus enters into a dormant state, whereas the characteristics of the HTLV-I related diseases indicate that their genesis requires activation of the dormant virus by a Tax-independent mechanism. In the present study we demonstrate that a variety of stress-inducing agents (TPA, cisplatin, etoposide, taxol, and 3-methylcholanthrene) are capable of Tax-independent activation of HTLV-I LTR and that this activation is detected mainly in cells that are undergoing through the apoptotic process. Furthermore, it is demonstrated that both apoptosis induction and HTLV-I LTR activation are inhibited by Bcl-2 and by PKC, indicating that these two processes are mechanistically cross-linked. In addition, using an HTLV-I producing human T-cell line which permanently express the negatively transdominant tax mutant, Delta58tax, under the Tet-Off control system, we prove that the virally encoded Tax protein protects the host cells from apoptosis. Together, these data suggest that activation of the dormant virus in the carriers' infected T-cells by certain stress-inducing conditions and protecting these cells from the consequent apoptotic death by the viral Tax protein emerging after this activation, might be the basis for switching the virus from latency to a pathogenic phase.

Specific interaction of a novel foamy virus Env leader protein with the N-terminal Gag domain.

Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa.

Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C.

We have previously demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) activates human T-cell leukemia virus type-I long terminal repeat (LTR) in Jurkat cells by a protein kinase C (PKC)-independent mechanism involving a posttranslational activation of Sp1 binding to an Sp1 site located within the Ets responsive region-1 (ERR-1). By employing the PKC inhibitor, bisindolylmaleimide I and cotransfecting the reporter LTR construct with a vector expressing PKC-alpha, we demonstrated, in the present study, that this effect of TPA was not only independent of, but actually antagonized by, PKC. Electrophoretic mobility shift assays together with antibody-mediated supershift and immuno-coprecipitation analyses, revealed that the posttranslational activation of Sp1 was exerted by inducing the formation of Sp1-p53 heterocomplex capable of binding to the Sp1 site in ERR-1. Furthermore, we demonstrated that Jurkat cells contain both wild-type (w.t.) and mutant forms of p53 and we detected both of them in this complex at variable combinations; some molecules of the complex contained either the w.t. or the mutant p53 separately, whereas others contained the two of them together. Finally, we showed that the Sp1-p53 complexes could bind also to an Sp1 site present in the promoter of another gene such as the cyclin-dependent kinase inhibitor p21(WAF-1), but not to consensus recognition sequences of the w.t. p53. Therefore, we speculate that there might be several other PKC-independent biological effects of TPA which result from interaction of such Sp1-p53 complexes with Sp1 recognition sites residing in the promoters of a wide variety of cellular and viral genes.

Characterization of peptide substrates and viral enzyme that affect the cleavage site specificity of the human spumaretrovirus proteinase.

Oligopeptides that correspond to proteolytic cleavage site junctions of the native Gag and Pol proteins are specifically cleaved by retroviral aspartate proteases (PRs). The role of the flap subdomain of the PR of the human spumaretrovirus (HSRV) and of substrate peptides in cleavage site specificity was analyzed by site-directed mutagenesis. Native and mutant peptides were subjected to proteolysis by the authentic and mutated recombinant viral enzyme. The results reveal that Glu residue 54 of the HSRV PR is an essential specificity determinant for proteolytic processing of the structural proteins. Peptides that represent in vivo cleavage sites were susceptible to proteolysis by the recombinant HSRV PR, but one peptide located at the junction between the PR and reverse transcriptase domains was completely resistant to cleavage. Thus the data indicate that a proteolytic cleavage between these domains does not occur in vivo. Naturally occurring and mutant forms of the cleavage-resistant peptide were therefore analyzed by circular dichroism to determine if differences existed in the secondary structures of the peptides that did or did not serve as substrates. The data show that differences in the secondary structure of the native and mutant peptides analyzed does not seem to play a crucial role for cleavage site specificity in HSRV PR. Instead highly conserved hydrophobic residues at distinct positions of the HSRV cleavage site junctions contribute to the specificity observed as reported for HIV-1 PR.

Human foamy virus Bel 1 transactivator/estrogen receptor fusion proteins allow inducible transactivation of both human foamy virus promoters.

Recombinant plasmids that express human foamy virus (HFV) Bel 1 transactivator and human estrogen receptor (ER) fusion proteins were constructed. The HFV bel 1 gene was inserted up- and downstream of the ER gene. Recombinant Bel 1-ER and ER-Bel 1 fusion proteins were expressed in eukaryotic cells. In the absence of estrogen, the ER moiety of the fusion proteins suppressed Bel 1-mediated transactivation as measured in CAT reporter gene-based transactivation assays. However, transactivation of the HFV LTR and the HFV internal promoter by Bel 1-ER and ER-Bel 1 fusion proteins was recovered in the presence of estrogen. Thus, the transactivation function of the Bel 1 moiety of the chimeric Bel 1-ER fusion proteins can be efficiently, specifically, and intentionally activated and inactivated by simply adding the low-molecular weight effector estrogen.

Characterization of the humoral immune response and virus replication in cats experimentally infected with feline foamy virus.

Cats were experimentally infected with cell culture-adapted feline foamy virus (FFV, spumaretrovirinae) isolate FUV. FFV was consistently recovered from peripheral blood leukocytes and throat samples of FFV-infected cats starting 2 to 3 weeks postinfection (p. i.), indicative of the establishment of persistent FFV infections. Viral persistence was established, even despite neutralizing antibodies that appeared early after infection. The humoral immune response toward FFV was quantitatively and qualitatively analyzed over time. FFV Gag-specific antibodies were first detected 2 weeks p. i. and increased further; reactivities to the other structural and nonstructural FFV proteins appeared slightly delayed. Reactivities against FFV Pol and Gag proteins were detectable by immunoblotting and radioimmunoprecipitation, whereas the latter techniques had to be employed for the unambiguous detection of FFV Env-, Bet-, and Bel 1-specific antibodies.

Induction of cellular genes is mediated by the Bel1 transactivator in foamy virus-infected human cells.

To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.

The intact retroviral Env glycoprotein of human foamy virus is a trimer.

Electron microscopy of negatively stained human foamy virus particles provides direct evidence for the trimeric nature of intact Env surface glycoproteins. Three-dimensional image reconstruction reveals that the Env trimer is a tapering spike 14 nm in length. The spikes were often arranged in hexagonal rings which shared adjacent Env trimers.

Construction of infectious feline foamy virus genomes: cat antisera do not cross-neutralize feline foamy virus chimera with serotype-specific Env sequences.

Full-length genomes of the feline foamy virus (FFV or FeFV) isolate FUV were constructed. DNA clone pFeFV-7 stably directed the expression of infectious FFV progeny virus indistinguishable from wild-type, uncloned FFV isolate FUV. The env and bel 1 genes of pFeFV-7 were substituted for by corresponding sequences of the FFV serotype 951 since previous studies implicated a defined part of FFV Env protein as responsible for serotype-specific differences in serum neutralization (I. G. Winkler, R. M. Flügel, M. Löchelt, and R. L. P. Flower, 1998. Virology 247: 144-151). Recombinant virus derived from chimeric plasmid pFeFV-7/951 containing the hybrid env gene and the parental clone pFeFV-7 were used for neutralization studies. By means of a rapid titration assay for FFV infectivity, we show that progeny virus derived from plasmid pFeFV-7 was neutralized by FUV- but not by 951-specific antisera, whereas pFeFV-7/951-derived chimeric virus was neutralized by 951-specific antisera only. Both recombinant proviruses will be useful for repeated delivery of foreign genes for therapeutic gene applications into cats.

Molecular characterization of proteolytic processing of the Gag proteins of human spumavirus.

Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.

Nuclear localization of the functional Bel 1 transactivator but not of the gag proteins of the feline foamy virus.

Interactions between host cells and foamy or spumaretroviruses are different from those of other known retroviruses. Previous work has suggested that the Gag and high-affinity DNA-binding Bel 1 transactivator of human foamy virus are localized in the nuclei of infected cells. Using two independent detection methods, we show here that the functionally active Bel 1 transactivator protein of feline foamy virus is of nuclear localization. In contrast to that reported for the human foamy virus Gag protein, the cat foamy virus Gag proteins exclusively localized in the cytoplasm close to perinuclear regions.

Detection and molecular characterisation of feline foamy virus serotypes in naturally infected cats.

The characterisation of two distinct feline foamy virus sequence groupings in the external surface portion of the viral env gene are reported. Although amino acid identities in the Gag nucleocapsid, Pol protease, and Env transmembrane domains were greater than 92% in the 12 proviral sequences examined, two distinct sequence groups were observed in the Env surface (SU) protein. Only 57% amino acid identity was observed in the Env SU between the two groups designated FUV7-like or 951-like, while within these groups > 97% identity was found. Isolates FUV7 and 951 represent two serogroups previously characterised by Flower et al. (1985). A 100% correlation was found among FeFV seroreactivity, virus isolation, and detection of viral DNA in feline leucocytes using a single round of PCR amplification. Serum neutralisation assay using autologous virus, as well as isolates 951 and FUV7, revealed that viruses with FUV7-like sequences were in a single neutralisation group and viruses with 951-like sequences were in a single neutralisation group. Based on these results, group-specific PCRs were developed, using the same sense primer with an antisense primer specific for each group. Using he PCR, no evidence of superinfection of any cat with virus from both serogroups was detected.

Molecular characterization of proteolytic processing of the Pol proteins of human foamy virus reveals novel features of the viral protease.

Spumaviruses, or foamy viruses, express a pol-specific transcript that codes for a Pol polyprotein that consists of the protease, reverse transcriptase, ribonuclease H, and the integrase domains. To delineate the proteolytic cleavage sites between the Pol subdomains, recombinant human foamy virus (HFV) Pol proteins were expressed, purified by affinity chromatography, and subjected to either HFV protease assays or autocatalytic processing. In control experiments, HFV protease-deficient mutant proteins in which the active site Asp was replaced by an Ala residue were used to rule out unspecific processing by nonviral proteases. Specific proteolytic cleavage products were isolated, and the cleavage sites were analyzed by amino acid sequencing. Peptides spanning the resulting cleavage sites were chemically synthesized and assayed with HFV protease, and the cleaved peptides were subjected to mass spectrometry. The cleavage site sequences obtained were in complete agreement with the amino-terminal sequences from amino acid sequencing of authentic cleavage products of the HFV Pol proteins. Analysis by fast-protein liquid chromatography of a short version of the active HFV protease revealed that the enzyme predominantly formed dimeric molecules.

The carboxy-terminal p3Gag domain of the human foamy virus Gag precursor is required for efficient virus infectivity.

Proteolytic processing of foamy virus Gag proteins appears to be different from that of other retroviruses. A single carboxy-terminal cleavage site is consistently detectable in human foamy virus (HFV) Gag precursor protein p74Gag derived from infected cells and/or purified virus particles. Using a recombinant HFV protease, we have determined the p74Gag cleavage site that results in p70Gag and the carboxy-terminal p3Gag (Pfrepper et al., 1997, Biochem. Biophys. Res. Commun. 237, 548-553). To study the biological functions of p3Gag, proviral DNA clones were constructed coding for a carboxy-terminally truncated p70Gag lacking the entire p3Gag protein. Removal of p3Gag resulted in an about 100-fold lower virus titer. The expression of other HFV proteins and the processing of Pol proteins were indistinguishable from those of wild-type-transfected cells. The defect in viral infectivity of the p3 mutants was partially restored by coexpressing the full-length p74Gag protein in trans. The deletion of p3Gag resulted in particle assembly with wild-type virion morphology and encapsidation of Pol proteins. Our data show that the carboxy-terminal p3Gag protein has an important function for viral infectivity but is not required for preassembly of capsids, virus morphogenesis, and incorporation of Pol proteins into virions.

Characterization of the genome of feline foamy virus and its proteins shows distinct features different from those of primate spumaviruses.

The genome of the feline foamy virus (FeFV) isolate FUV was characterized by molecular cloning and nucleotide sequence analysis of subgenomic proviral DNA. The overall genetic organization of FeFV and protein sequence comparisons of different FeFV genes with their counterparts from other known foamy viruses confirm that FeFV is a complex foamy virus. However, significant differences exist when FeFV is compared with primate foamy viruses. The FeFV Gag protein is smaller than that of the primate spumaviruses, mainly due to additional MA/CA sequences characteristic of the primate viruses only. Gag protein sequence motifs of the NC domain of primate foamy viruses assumed to be involved in genome encapsidation are not conserved in FeFV. FeFV Gag and Pol proteins were detected with monospecific antisera directed against Gag and Pol domains of the human foamy virus and with antisera from naturally infected cats. Proteolytic processing of the FeFV Gag precursor was incomplete, whereas more efficient proteolytic cleavage of the pre125Pro-Pol protein was observed. The active center of the FeFV protease contains a Gln that replaces an invariant Gly residue at this position in other retroviral proteases. Functional studies on FeFV gene expression directed by the promoter of the long terminal repeat showed that FeFV gene expression was strongly activated by the Bell/Tas transactivator protein. The FeFV Bell/Tas transactivator is about one-third smaller than its counterpart of primate spumaviruses. This difference is also reflected by a limited sequence similarity and only a moderate conservation of structural motifs of the different foamy virus transactivators analyzed.

Expression and molecular characterization of an enzymatically active recombinant human spumaretrovirus protease.

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 the E. coli trxA protein, 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.

A rapid streptavidin-capture ELISA specific for the detection of antibodies to feline foamy virus.

We report a simple procedure for the rapid development of an ELISA with the potential for wide application to any defined protein antigen. The procedure involves the expression of protein encoded by a PCR product, using a commercially available T-vector that adds a biotin tag, and a single step purification by affinity for streptavidin for direct use in ELISA. In our experiments, a recombinant protein from the nucleocapsid domain of the feline foamy virus gag gene was expressed as a fusion protein with a biotin tag and then applied directly to streptavidin-coated ELISA wells. An extract from a clone with the insert in antisense orientation was used as a control. Non-specific reactions with antigen extracts from both sense and antisense clones were observed in 6 of the 376 (1.6%) sera tested. Antibody to feline foamy virus, which forms a stable persistent infection in cats, was detected in 107 of 201 (53%) Australian cats, but none of 175 sera from veterinarians. There was a 100% correlation between FeFV antibody detected by ELISA, immunoblot, serum neutralisation and virus isolation, confirming that this test is sensitive and specific.

The long terminal repeats of human immunodeficiency virus type-1 and human T-cell leukemia virus type-I are activated by 12-O-tetradecanoylphorbol-13-acetate through different pathways.

The LTRs of HIV-1 and HTLV-I have been shown by several laboratories to be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). This agent is a potent activator of protein kinase C (PKC). However, long exposure to TPA downregulates PKC in many cell types. We demonstrated that TPA treatment of Jurkat cells for more than 24 hr resulted in a sever depletion of this enzyme. Therefore, to explore the role of PKC in the effect of TPA on these LTRs, we transfected Jurkat cells with HIV-1 LTR-CAT or HTLV-I LTR-CAT construct after 72 hr of TPA pretreatment. While this TPA pretreatment considerably reduced the HIV-1 LTR basal expression, it strongly stimulated the expression of HTLV-I LTR. Furthermore, when TPA was added after transfection, a strong stimulation of HIV-1 LTR was observed, which could be abrogated by PKC inhibitors like H7 and chelerythryn. However, under these conditions TPA stimulated HTLV-I LTR to a lesser extent than did the long-term TPA pretreatment. Moreover, this stimulation was enhanced by the PKC inhibitors. Thus our data indicate that while the effect of TPA on HIV-1 LTR is strictly dependent on PKC activity, its effect on HTLV-I LTR is exerted via a different pathway that not only does not require PKC activation but rather seems to be antagonized by the activated PKC. Using a deletion mutant of HTLV-I LTR we mapped the PKC-independent effect of TPA to the c-ets responsive region 1 (ERR-1) located in U3 of this LTR.