Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors.

As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are devoid of almost the complete env gene plus the internal promoter - accessory bel gene cassette including the gene for the viral transcriptional transactivator Bel1/Tas. In these Bel1/Tas-independent vectors, expression of the lacZ (beta-galactosidase) marker gene is directed by the heterologous, constitutively active human ubiquitin C promoter (ubi). Env-transcomplemented vectors have un-concentrated titers of more than 10(5) transducing units/ml. The vectors allow efficient transduction of a broad array of diverse target cells, which can be increased by repeated vector exposure. However, the number of lacZ marker gene expressing cells decreased slightly upon serial passages of the transduced cells. Vectors carrying a self-inactivating (SIN) deletion of the TATA box and most parts of the viral promoter were not rescued by wt FFV whereas those with the intact or a partially deleted promoter were readily reactivated. This finding indicates that the viral promoters are in fact non-functional, pointing to a highly advantageous safety profile of these new FFV-ubi-lacZ-SIN vectors.

Kinetics and characteristics of replication-competent revertants derived from self-inactivating foamy virus vectors.

In this study, self-inactivating (SIN) retroviral vectors based on feline foamy virus (FFV) were constructed and analysed. The FFV SIN vectors were devoid of the core FFV long terminal repeat promoter plus upstream sequences but contained all structural and regulatory genes. This design allowed sensitive detection of replication-competent revertants (RCRs). The FFV SIN vectors efficiently transduced the green fluorescence protein into recipient cells. However, RCRs appeared after serial passages of transduced cells. In all RCR clones analysed, parts of the heterologous cytomegalovirus immediate early promoter, originally driving expression of the FFV vector genome, were taken up to restore the deleted SIN promoter function required for replication competence. The RCRs were strongly reduced in replication capacity compared with the parental replication-competent vectors containing the FFV promoter. In all RCR genomes analysed, the uptake of the heterologous promoter was accompanied by deletion of almost the complete marker gene. Although the RCRs described in this study may not have the capacity to spread in humans and animals, they may pose a theoretical risk, for instance during transduction of haematopoietic stem cells. Thus, FV-based SIN vectors require additional genetic modifications in order to avoid RCRs.

Potential of zoonotic transmission of non-primate foamy viruses to humans.

The zoonotic introduction of an animal pathogen into the human population and the subsequent extension or alteration of its host range leading to the successful maintenance of the corresponding pathogen by human-to-human transmission pose a serious risk for world-wide health care. Such a scenario occurred for instance by the introduction of simian immunodeficiency viruses into the human population resulting in the human immunodeficiency viruses (HIV) and the subsequent AIDS pandemic or the proposed recent host range switch of the SARS coronavirus from a presently unknown animal species to humans. The occurrence of zoonotic transmissions of animal viruses to humans is a permanent threat to human health and is even increased by changes in the human lifestyle. In this review, the potential of the zoonotic transmission of bovine, feline and equine foamy retroviruses will be discussed in the light of well-documented cases of zoonotic transmissions of different simian foamy viruses to humans.

Demonstration of feline foamy virus in experimentally infected cats by immunohistochemistry.

Foamy viruses (FV) are complex retroviruses which are commonly isolated from cats, cattle and non-human primates. The infection is persistent and infected animals have a sustained antibody response. The role of FV in diseases remains unclear, in cats, a possible association with uncharacterized renal symptoms remains to be confirmed. To demonstrate feline FV (FFV) in tissues of experimentally infected cats three polyclonal monospecific antisera from rabbits against three different viral proteins, the structural Gag and the non-structural Bel 1 and Bet proteins were tested for their applicability in immunohistochemistry with paraffin sections. Only the Bet antiserum allowed detection of FFV-specific proteins, the antibodies against Gag and Bel 1 did not work even after pre-treatment of the slides with proteinase K or cooking in a pressure cooking pot. The Bet-reactive antibodies were detected using a commercial streptavidin kit and revealed Bet in the cytoplasm of cells from different lymphoid tissues like lymphnodes, tonsils, thymus and spleen. The method described opens new ways to explore the in vivo replication and tissue specificity of FFV and its possible role in disease.

Foamy virus transactivation and gene expression.

An overview of the pattern and mechanisms of spuma or foamy virus (FV) gene expression is presented. FVs are complex retroviruses with respect to their genetic outfit and the elements used to control and regulate expression of the viral genome. The increased insight into transcriptional and posttranscriptional mechanisms has revealed that the FVs are distinct, unconventional retroviruses clearly apart from the orthoretroviruses. Although less characterized than the orthoretroviruses, FVs have several unique features that are important for construction and assembly of FV-based vectors for targeted gene delivery and vaccination purposes. Some of these distinguishing features are directly related to the FV-specific mechanisms of gene expression and include (1) the presence of an internal, functional active second transcription unit for expression of the nonstructural genes, (2) the utilization of a subgenomic, spliced transcript for Pol protein expression, and (3) distinct but not yet understood mechanisms for the nuclear exit of defined transcripts and thus an additional level of posttranscriptional control of gene expression. Finally, the interactions of the viral transactivator not only with both viral promoters but also with regulatory elements controlling the expression of defined cellular genes are an important issue with respect to vector development and the apparent apathogenicity of FVs in their natural hosts.

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.

The bet gene of feline foamy virus is required for virus replication.

Foamy viruses (FV) are complex retroviruses with additional bel genes located between env and the 3' long-terminal repeat. The functions of the bel 2 and bet genes are unknown and both are dispensable for replication of the prototypic human foamy virus in cell cultures. We examined the function(s) of bel 2 and bet of the distantly related feline foamy virus (FFV) in the proviral context. Mutagenesis was used to alter the Bel 2 and Bet or to abrogate their expression. The Bel 2/Bet mutants showed a 1000-fold reduced viral titer in feline kidney cells; in human 293T cells, viral titer was only about 10-fold reduced compared to wild-type FFV. In both cell types, the Bel 2/Bet mutations resulted in a reduced release of FFV particles. The results indicate that FFV Bet is required for efficient virus replication. The functions of the Bel 2 and Bet proteins are discussed.

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.

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.

Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses.

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 AUG461 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.

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.

Epidemiology of feline foamy virus and feline immunodeficiency virus infections in domestic and feral cats: a seroepidemiological study.

Although foamy viruses (Spumaviruses) have repeatedly been isolated from both healthy and diseased cats, cattle, and primates, the primary mode of transmission of those common viruses remains undefined. A database of the feline foamy virus (FeFV) and feline immunodeficiency virus (FIV) antibody status, age, and sex of 389 domestic cats presented to veterinarians was assembled. A similar database for 66 feral (wild) cats was also assembled. That FeFV antibody status reflects infection was validated by PCR. Both FeFV and FIV infection rates were found to gradually increase with age, and over 70% of cats older than 9 years were seropositive for FeFV. In domestic cats, the prevalence of FeFV infection was similar in both sexes. In feral cats, FeFV infection was more prevalent in female cats than in male cats. Although both FeFV and FIV have been reported to be transmitted by biting, the patterns of infection observed are more consistent with an interpretation that transmission of these two retroviruses is not the same. The prevalence of FIV infection is highest in nondesexed male cats, the animals most likely to display aggressive behavior. The gradual increase in the proportion of FeFV-infected animals is consistent with transmission of foamy viruses by intimate social contact between animals and less commonly by aggressive behavior.

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.