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In contrast to other retroviruses, foamy viruses (FVs) generate their Pol protein precursor independently of the Gag protein from a spliced mRNA. The exact mechanism of Pol protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells.

Foamy viruses (FVs) harbour a transcriptional transactivator (Tas) and two Tas-responsive promoter regions, one in the 5′ long terminal repeat (LTR) and the other an internal promoter (IP) in the envelope gene. To analyse the mechanism of transactivation of the FVs, the specificity of feline FV (FFV) Tas protein, which is more distantly related to the respective proteins of non-human primate origin, were investigated. FFV Tas has been shown specifically to activate gene expression from the cognate promoters. No cross-transactivation was noted of the prototype foamy virus and human immunodeficiency virus type 1 LTR. The putative transactivation response element of FFV Tas was mapped to the 5′ LTR U3 region (approximately nt −228 to −195). FFV Tas binds to this element in addition to a previously described sequence (position −66 to −51). It is therefore concluded that FFV Tas is a DNA-binding transactivator that interacts with at least two regions in the virus LTR.

Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127Pol, p85RT/RN and p40IN served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.

Foamy viruses (FVs) are complex retroviruses which require for their replication the activity of a transcriptional trans-activator (Tas) as well as Tas-responsive elements in the viral promoters. A mutant of the chimpanzee FV strain, CFV/hu (previously called human FV), genome in which most of the U3 promoter of the CFV long terminal repeat was substituted by the constitutively active human cytomegalovirus immediate early gene enhancer/promoter was constructed. This plasmid (pTS12) and a derivative (pTS13), which has a deletion in the tas gene, gave rise to replication-competent virus. Compared with parental CFV, both mutants replicated only very poorly, with retarded growth kinetics and maximal cell-free virus titres reduced by approximately three orders of magnitude. Mutation of the DD35E motif of the CFV integrase to DA35E rendered the recombinant TS virus replication-deficient. This indicated that provirus integration is probably still required for this FV derivative, which had been converted from a complex regulated retrovirus into a simple one by incorporation of a constitutively active promoter from another virus which regularly does not integrate into the host cell genome.

Foamy viruses (FVs) make use of a replication strategy which is unique among retroviruses and shows analogies to hepadnaviruses. The presence of an integrase (IN) and obligate provirus integration distinguish retroviruses from hepadnaviruses. To clarify whether a functional IN is required for FV replication, a mutant in the highly conserved DD35E motif of the active centre was analysed. This mutant was found to be able to express Gag and Pol protein precursors and cleavage products and to generate and deliver cDNA. However, this mutant was replication-deficient. The junctions of individual foamy proviruses with cellular DNA were sequenced. The findings suggest that FV integration is asymmetrical, because the proviruses started with what is believed to be the U3 end of the free linear DNA to generate the conventional TG dinucleotide, while apparently two nucleotides from the U5 end were cleaved to create the complementary CA dinucleotide. Alignment of known FV genome sequences indicated that this mechanism of integration is not restricted to the two FV isolates from which integrates were studied, but appears to be a common feature of this retrovirus subfamily. In conclusion, with respect to the necessity of a functionally active IN for virus replication FVs behave like other retroviruses; their mechanism of integration, however, is probably unique.

The foamy viruses (FVs) are a genus of complex retroviruses that has recently been found to possess several novel molecular features. There is increasing interest in the development of FVs as novel vectors for gene delivery. As there are remarkably few published studies of FV proteins, these recent findings prompted us to predict the structural features of FV glycoproteins with the aid of computer programs. We analysed all seven available FV Env sequences, a greater number of sequences than in previously published analyses. The relative rates of change for FV structural proteins were Pol < Env < Gag in increasing order, which differs from all other retroviruses. We determined that this difference is primarily caused by a higher relative rate of change for FV Gag proteins. We analysed the functional domains of FV glycoproteins and found that their structural organization was generally similar to other retroviruses. Putative structures were identified for the signal peptide, cleavage site, fusion peptide, membrane-spanning domain and the unique endoplasmic reticulum retrieval signal. Based on the predicted secondary structure of the transmembrane glycoprotein (TM) subunit, gp47, we also identified a unique prolonged central 'sheets and loops' region as the dominant feature of an unusually lengthy TM ectodomain. This lengthy central domain was flanked at each end by alpha-helices. The predictions reported here will stimulate and facilitate experimental approaches to better understand the structure and function of FV glycoproteins, and should assist in the planning and development of FV vectors.

A mouse model was developed to study the virus-host interaction of molecularly cloned human foamy virus (HFV) in vivo. The infectious process was analysed in two mouse strains, CBA/Ca and C57BL/6J, over a period of 24 weeks by PCR on DNAs from various animal tissues; virus serology was examined by immunoblotting. The infection persisted in both mouse strains and did not induce clinical symptoms. Upon infection of adult CBA/Ca mice HFV became detectable by PCR in an increasing number of organs over time. In contrast, in C57BL/6J mice, after an initial phase of dissemination, viral DNA sequences were found only in a few organs. Interestingly, the different course of infection was accompanied by differences in the antiviral immune response. In particular, C57BL/6J mice were high responders with respect to antibodies to the viral Bet protein, while CBA/Ca mice were low responders.

We examined the effect of human foamy virus (HFV) infection on the expression of human major histocompatibility complex molecules. Our data show that in vitro HFV infection of U373-MG glioblastoma cells results in increased expression of class I human leukocyte antigen (HLA) and transcripts. Transient transfection assays of plasmids containing the reporter gene chloramphenicol acetyl transferase driven by different 5' deletions of the HLA-A11 class I promoter allowed identification of cis-acting elements involved in this regulation. HFV infection has two opposite effects on the HLA class I promoter: transactivation of the HLA-A11 promoter through a positive regulatory element located in the -525 to -335 region upstream of exon 1 and down-regulation of transcriptional activity driven by the -335 to -205 class I promoter region. Additional experimental data indicate that the effect of HFV on HLA class I expression is not mediated by the interferon pathway.

In order to establish criteria for the Serodiagnosis of foamy virus infections we investigated the extent to which sera from iofected 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. Rahbit 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 Mrican green monkey origin. This was reßected 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 o/o) showed antiborlies against the Bet protein, indicating that Bet antigen is of value in sero1ogical screening for foamy virus infections.

The nucleotide sequence of bovine respiratory syncytial virus (RSV), ATCC strain A51908 fusion (F) glycoprotein gene cDNA was determined. The amino acid sequence deduced was then compared to those of two different isolates of bovine RSV, strains RB 94 and 391-2, and the A and B subtypes of human RSV, strains 18537 and A2. The bovine RSV F protein is highly conserved between the three isolates, A51908 has 97% amino acid identity to RB 94, and 99% identity to 391-2. The F proteins of both the A and B types of human RSV are 81% identical to that of A51908. The cDNA clone was expressed using a baculovirus vector and the expressed recombinant F protein produced in SF9 cells was characterized by Western blot analysis. The recombinant F protein was post-translationally cleaved into the active form and reacted with serum from bovine RSV-infected calves.

The nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94% identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features of BRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43% amino acid identity. As described for HRSV, the 3'-terminal end of the M2 mRNA overlaps with the 5' end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75%. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.

We recently reported the presence of linear duplex DNA intermediates with a gap in the middle of the molecules in the replicative cycle of human (HSRV) and simian (SFV1) spumaviruses. The polypurine tract (PPT), at the 5' boundary of the 3' long terminal repeat, was found to be duplicated in the gap region. By molecular analysis of HSRV proviral DNA with region- and strand-specific probes, we have now determined that the gap is located on plus-strand DNA and that it is 120 bases long with the 3' end mapping at the duplicated PPT site. Kinetic analysis of proviral DNA provided evidence that the gap did not result from processing of a complete, full-length DNA molecule. These data strongly suggest that plus-strand DNA synthesis is initiated at both PPT sites.

Summary A persistent foamy virus infection was established in rabbits and its effect on the cell-mediated (CMI) and humoral immune response was studied. Virus was consistently isolated from viable, peripheral blood mononuclear cells collected 7 to 164 days after inoculation, by co-cultivation, with cell monolayers. The response of leukocytes from infected rabbits to in vitro stimulation with phytohaemagglutinin was studied by two techniques: incorporation of 3H-thymidine and production of the lymphokine, immune interferon. Both parameters of the cell-mediated immune response were depressed in leukocytes collected from foamy virus infected rabbits during the first 2 weeks of the infection. This depression in the cell-mediated immune response was not observed after 14 days p.i. Since primary or reactivation infections with herpes viruses are common following immunosuppression, it was interesting to note that one of the rabbits persistently infected with foamy virus developed a herpes virus infection. The humoral response in infected rabbits appears to be unaffected since the virus infection did not alter the development of antibodies to sheep erythrocytes. Transient depression of the cell-mediated immune response in foamy virus infections may be important in initiating persistence and demonstrates for the first time that foamy viruses can, indeed, have adverse effects.

Preparations of highly concentrated simian foamy virus type 1 (SFV1)agglutinate guinea pig red blood cells. The agglutination occurs both at 22 degrees and 37 degrees C. It is inhibited by specific antisera against SFV1 but also by antisera prepared against several other types of simian foamy viruses.

Summary The antigens from cytolytic infections of HEp2 cells by type I simian foamy virus produced two multicomponent precipitation lines when tested by immunodiffusion with the homologous hyperimmune rabbit antiserum. The antigens obtained from a non-productive infection of MK5 virus in HEp2 cells produced only those precipitation lines which corresponded with the inner lines obtained from the cytolytic infection. Similarly, hyperimmune rabbit antiserum against antigens extracted from the persistent infection lacked the antibody which was responsible for the outer lines of precipitation. Indirect immunofluorescence with acetone-fixed and unfixed cells using the homologous and heterologous sera confirmed the absence of antigens in the persistent infection and showed that an antigen is produced in the persistently infected cells which is either absent or present in very small amounts in cytolytically infected cells. Neutralization experiments and ether treatment suggested that the missing antigens in the persistent infection were the envelope components of foamy virus. It is proposed that the persistent infection has properties in common with some infections by RNA tumour viruses.

The maturation of feline syncytium-forming virus (FSFV), a member of the foamy virus sub-family (Spumavirinae), has been studied by electron microscopy of thin sections of infected feline embryo (FEA) cells. The initial event observed was formation of crescent-shaped nucleoids at the plasma membrane. As budding progressed, the nucleoid became circular in outline with an electron-lucent centre in fully mature extracellular particles. These observations suggested that the maturation of FSFV in fully permissive FEA cells resembled that of C-type RNA tumour viruses, rather thant the B-type mouse mammary tumour virus. In this respect FSFV may be distinct from other foamy viruses. However, like other foamy viruses FSFV possessed reverse transcriptase activity. Polymerase activity co-sedimented with infectivity in an equilibrium density gradient and exhibited a preference for poly(rA).oligo(dT)10 over poly(dA).oligo(dT)10 as exogenous template.

Summary DNA isolated from cells infected with FSFV (a foamy virus) is infectious when tested on susceptible cells. The virus produced by this infectious DNA is identical to the original infecting virus in terms of plaque and virion morphology and serology.