Characterization of a cis-Acting Sequence in the pol Region Required To Transfer Human Foamy Virus Vectors (original) (raw)
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Sequences in pol Are Required for Transfer of Human Foamy Virus-Based Vectors
1998
A series of vectors with heterologous genes was constructed from HSRV1, an infectious clone of human foamy virus (HFV), and transfected into baby hamster kidney cells to generate stably transfected vector cell lines. Two cis-acting sequences were required to achieve efficient rescue by helper virus. The first element was located at the 5* end upstream of position 1274 of the
Identification of pol-Related Gene Products of Human Foamy Virus
Virology, 1993
Human foamy viruspol gene fragments were molecularly cloned into a procaryotic expression vector. The expression pattern of the cloned fragments and nucleotide sequence analysis of the 5' pol gene region revealed that in HFV the protease (PR) is located in the pol open reading frame. Purified recombinant proteins were used to generate antibodies in rats. ln immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (M,) of 127K was identified by antibodies directed against the reverse transcriptase (RT), RNaseH, or integrase (IN) domeins of pol. With concentrated virus as antigen, the RT and RNaseH antibodies recognized a protein of 80K, the IN antiserum recognized a protein of 40K, and the PR antiserum detected a protein of approximately 10K.
Journal of Virology, 2000
. Two PFV cis-acting sequences have been mapped in the 5 region of the RNA (pre-)genome and in the 3 pol genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5 untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5 UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV gag gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and the gag gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentialy packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the pol gene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve.
Journal of Virology, 2001
It has been suggested that sequences located within the 5 noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete ϳ151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5 noncoding region but that is inactive when inverted or when placed in the 3 noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5 splice site positioned at ؉51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3 to this splice site. Analysis of total cellular gag and pol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave the pol 5 splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5 noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3 noncoding sequence. Neither the R element nor the introduced 5 intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in cis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3 noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847-4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.
Generation of an improved foamy virus vector by dissection of cis-acting sequences
Journal of General Virology, 2009
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. 127 kDa Pol precusor protein is encapsidated, the cleavage products of 85 (PR-RT) and 40 kDa IN) are not packaged separately (Peters et al., 2005). This implicates cleavage of the Pol precursor to occur after encapsidation. Within the CAS elements the sequence required for Pol encapsidation has been identified (Peters et al., 2005). (iii) Within the 39
Properties of human foamy virus relevant to its development as a vector for gene therapy
Journal of General Virology, 1999
The Spumaviridae (foamy viruses) are increasingly being considered as potential vectors for gene therapy, yet little has been documented of their basic cell biology. This study demonstrates that human foamy virus (HFV) has a broad tropism and that the receptor for HFV is expressed not only on many mammalian, but on avian and reptilian cells. Receptor interference assays using an envelope-expressing cell line and a vesicular stomatitis virus/HFV pseudotype virus demonstrate that the cellular receptor is common to all primate members of the genus. The majority of foamy virus particles assemble and remain sequestered intracellularly. A rapid and quantitative method of assaying foamy virus infectivity by reverse transcriptase activity facilitates the use of classical protocols to increase infectious virus titres in vitro to 10 6 TCID/ml. 0001-6313 # 1999 SGM CAAD Downloaded from www.microbiologyresearch.org by
Identification of Sites That Act Together to Direct Dimerization of Human Foamy Virus RNA in Vitro
Virology, 1997
Retroviral particles contain two molecules of genomic RNA, which are noncovalently linked near their 5 ends in a region called the dimer linkage structure (DLS). By using complementary DNA oligonucleotides and deletion mutants to impair RNA dimerization of the human foamy virus (HFV), three sites, designated SI, SII, and SIII, were found within a 159-nucleotide RNA fragment of HFV that are involved in dimerization in vitro. SI overlaps the primer-binding site; SII contains the palindromic sequence, UCCCUAGGGA, the disruption of which impairs dimer formation; and SIII extends into the gag gene. The first two sites are highly conserved in the other primate foamy viruses, SFV-1, SFV-3, and SFV cpz , whereas the third appears to be shared only by HFV and SFV cpz . RNA of HFV and SFV-3 could form heterodimers, indicating that both viruses dimerize by similar mechanisms. On testing thermal stability, dimers of the 159-nucleotide fragment dissociated between 40 and 70Њ, with half of the dimers dissociating at 55Њ. Since the splice donor site of HFV is located at position 51 of viral RNA, the DLS is part of the genomic RNA exclusively. ᭧ 1997 Academic Press
Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus
Nucleic Acids Research, 1995
Human foamy or spuma virus (HFV) codes for a distinct set of pol gene products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribonuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesls. The mutant gene products were bacterially expressed, purified by Ni 24 chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recomblnant HFV pol mutant proteins were characterised by in situ RT, RNase H and RNase H* assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-Hls. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while ONA polymerase activity increased 2-fold. A deletion of 11 amlno acid residues In the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant In the C-termlnal RH domain showed no RNase H but retained RNase H* activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
Cell-type-specific regulation of the two foamy virus promoters
Journal of virology, 2001
The foamy virus (FV) genome contains two promoters, the canonical long terminal repeat (LTR) promoter, containing three consensus AP-1 binding sites, and an internal promoter (IP) within the env gene. We investigated the regulation of the two promoters in lytic and persistent infections and found that in the presence of a constitutive source of the viral transactivator protein Tas, transactivation of the LTR promoter and that of the IP differ. In lytic infections, both the LTR promoter and the IP are efficiently transactivated by Tas, while in persistent infections, the IP is efficiently transactivated by Tas, but the LTR promoter is not.