An infectious and full-length molecular clone of genomic human spumaretrovirus (HSRV) DNA was constructed. The infectivity of the pHSRV 13 clone was demonstrated after transfection into susceptible ...cells by passage of HSRV-specific cytopathic effects as a cell-free culture supernatant, by electron microscopy of HSRV particles in pHSRV13 DNA-transfected cells, by detection of HSRV transcripts, and by identification of HSRV-encoded proteins with Env- and Bel-specific antisera in indirect immunofluorescence assays and in protein blotting. The predominant HSRV protein detected in immunoblots by both Bel 1- and Bel 2-specific antisera had an apparent molecular weight of 56 kDa and corresponds to Bet. The amino-terminus of Bet is encoded by part of a Bel 1-specific RNA and the larger Bet domain by an RNA species from the bel 2 gene (Muranyi, W., and Flu¨gel, R. M.
J. Virology 65, 727–735, 1991). HSRV-specific proteins of 36 and 43 kDa reacted with Bel 1 and Bel 2 antisera, consistent with the values calculated for the
bel 1 and
bel 2 gene products, respectively. Deletion mutagenesis of the transcriptional HSRV-specific
trans-activator
bel 1 and the
bet genes completely abolished the infectivity of the pHSRV13 clone. The defect in RNA, protein, and virion synthesis was
trans-complemented by cotransfection of an expression clone harboring the complete
bel coding region. This result demonstrates that the
bel 1 gene is required for viral replication. It remains to be determined whether other HSRV gene products, like
bet that share a common region with
bel 1, contributed to the defect observed.
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.
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 pre125(Pro-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 Bel1/Tas transactivator protein. The FeFV Bel1/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
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.
Retroviral vectors derived from foamy or spumaretroviruses are considered promising tools for targeted gene delivery and vaccination purposes. In order to fully exploit this potential, we identified ...essential
cis-acting sequences on the feline foamy virus (FFV) genome by constructing and analyzing a series of FFV-based replication-deficient vector genomes.
Cis-acting sequences essentially required for marker gene transfer were found to be localized at two sites on the FFV genome: (i) in the 5′-untranslated region and close to the
gag ATG and (ii) in the central part of the
pol gene. The presence of two
cis-acting sequences and their relative location on the FFV genome are similar but not identical to the functionally corresponding elements described for simian and primate foamy viruses.
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