Receptor targeting of adeno-associated virus vectors.

Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target AAV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the transmembrane and intracellular processing of this virus.

Replication of a foamy virus mutant with a constitutively active U3 promoter and deleted accessory genes.

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.

An active foamy virus integrase is required for virus replication.

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.

Characterization of a cis-acting sequence in the Pol region required to transfer human foamy virus vectors.

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5' gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3' pol region. Although small 5' and 3' deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.

Foamy virus particle formation.

Subgenomic expression plasmids for the so-called human foamy virus (HFV) structural gag, gag/pol, and env genes were constructed and used to analyze foamy virus particle formation by electron microscopy. Expression of an R-U5-gag-pol construct under control of the human cytomegalovirus immediate-early enhancer-promoter resulted in the formation of viral cores with a homogeneous size of approximately 50 nm located in the cytoplasm. Upon coexpression of an envelope construct, particles were observed budding into cytoplasmic vesicles and from the plasma membrane. Expression of the Gag protein precursor pr74 alone led to aberrantly formed viral particles of heterogeneous size and with open cores. Normal-shaped cores were seen after transfection of a construct expressing the p70gag cleavage product, indicating that p70gag is able to assemble into capsids. Coexpression of p70gag and Env resulted in budding virions, ruling out a requirement of the reverse transcriptase for capsid or virion formation. In sharp contrast to other retroviruses, the HFV cores did not spontaneously bud from cellular membranes. Radiochemical labeling followed by protein gel electrophoresis also revealed the intracellular retention of Env-deprived HFV capsids.

Human foamy virus reverse transcription that occurs late in the viral replication cycle.

Foamy viruses (FVs) are retroid viruses which use a replication strategy unlike those of other retroviruses and hepadnaviruses (S. F. Yu, D. N. Baldwin, S. R. Gwynn, S. Yendapilli, and M. L. Linial, Science 271:1579-1582, 1996). One of the striking differences between FVs and retroviruses is the presence of large amounts of linear genome-length DNA in FV-infected cells and in virions. We report here that large quantities of genome-length linear FV DNA accumulate in cells infected with FV, as determined by Southern blotting. To determine whether these unintegrated virus DNAs result solely from superinfection, we analyzed the occurrence of virus cDNA of the so-called human FV isolate (HFV) in cells transfected with a virus mutant deficient in the envelope gene and in cells which are resistant to superinfection due to stable expression of the envelope protein. We show that the synthesis of viral cDNA is independent of superinfection and that HFV synthesizes cDNA intracellularly as a late event in the replication cycle. To further confirm this finding, we performed inhibition studies with the reverse transcriptase inhibitor zidovudine (AZT). While AZT had no effect or only a minor effect on virus titers when added to cells prior to virus infection, viral titers were reduced by 3 or 4 orders of magnitude when the virus was produced from cells in the presence of AZT. Our results are most compatible with the hypothesis that the functional nucleic acid of the extracellular HFV consists of largely double-stranded linear DNA.

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked.

Characterization of human foamy virus proteins expressed by recombinant vaccinia viruses.

We report the generation of recombinant vaccinia viruses (VVs) expressing the gag, pol, bel-1, and bet open reading frames of human foamy virus (HFV), and the establishment of a transient, VV-T7 RNA polymerase-directed expression system for the HFV env gene. The correct expression of the HFV proteins was demonstrated by radioimmunoprecipitation using monospecific rabbit antisera, by analysis of the subcellular distribution (for VVgag, VVpol, VVbel-1, and VVbet), and by the ability to induce syncytium formation (for the env expression system). The HFV pol gene was successfully expressed using its own ATG start codon. Foamy viruses are regarded as retroviruses with intracytoplasmatic capsid assembly. However, when VVgag and VVpol were used to study the HFV Gag-Pol protein interaction and particle formation, no HFV capsid structures were observed in singly or doubly infected cells. In addition, no cleavage of the Pr74gag precursor molecule by the pol-encoded protease was detected in doubly infected cells. Our results indicate that foamy virus particle assembly is fundamentally different from that of other retroviruses.

Expression of human foamy virus reverse transcriptase involves a spliced pol mRNA.

In human foamy virus (HFV) the reverse transcriptase is expressed independently of the Gag protein as a 127-kDa Pol precursor molecule. Evaluating the mechanism of Pol expression we identified a spliced mRNA which uses the main 5' splice donor and a splice acceptor site located in the gag gene. The significance of this spliced transcript for HFV Pol expression was studied by constructing a virus with a mutated splice acceptor site. This virus was unable to express detectable Pol proteins after transient transfection. Replication of the mutant was studied by a sensitive assay based on HFV transactivator-stimulated expression of an integrated lacZ gene under control of the HFV long terminal repeat. Whereas in the first 2 weeks after transfection the mutant replicated 3 to 5 order of magnitude less well than wild-type virus, extracellular titers obtained thereafter were similar to those of wild-type virus. This increase in replication competence was accompanied by a reversion of the mutated splice acceptor site. The results underlined the importance of the spliced pol transcript for HFV replication and pointed to a second mechanism of Pol expression. Indicator gene assays suggest that this other mechanism is likely to be a transactivator-dependent cryptic promoter in the gag gene which gives rise to Pol-encoding transcripts.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses.

Nuclear degradation of nonsense mutated beta-globin mRNA: a post-transcriptional mechanism to protect heterozygotes from severe clinical manifestations of beta-thalassemia?

Nonsense mutations of the beta-globin gene are a common cause of beta-thalassemia. It is a hallmark of these mutations not only to cause a lack of protein synthesis but also a reduction of mRNA expression. Both the pathophysiologic significance and the underlying mechanisms for this surprising phenomenon have so far remained enigmatic. We report that the reduction of the fully spliced mutant beta-globin mRNA already manifests itself within the nucleus. In contrast, the levels of mutant pre-mRNA are normal. The promoter and the 5'-untranslated region (5'-UTR) of the herpes simplex virus type 1 thymidine kinase (HSV1 Tk) gene can independently circumvent this recognition/response mechanism in cis and restore nonsense mutated beta-globin mRNA expression to normal levels. These two genetic elements can thus exert a dominant influence on the post-transcriptional control of nonsense mutated beta-globin gene expression. While wild-type mRNA levels are restored by fusion of the HSV1 Tk 5'-UTR to the nonsense mutated beta-globin reading frame, translation of a wildtype reading frame in such a hybrid is precluded. In contrast, the HSV1 Tk promoter appears to efficiently deliver the mRNA to the translational apparatus. The 5'-UTR and the promoter sequences therefore control the nuclear fate of nonsense mutated beta-globin mRNA by separable pathways. The nuclear mRNA degradation mechanisms examined here may prevent the synthesis of C-terminally truncated beta-globin chain fragments and may protect heterozygotes from clinically relevant symptoms of beta-thalassemia.

Determination of mRNA fate by different RNA polymerase II promoters.

Translational stop mutations of the human beta-globin gene cause a reduction of cytoplasmic mRNA accumulation in thalassemia patients and in transfection models. The exact mechanism underlying this phenomenon has remained enigmatic but is known to be post-transcriptional. We have used transfected HeLa cells to study the expression of beta-globin mRNAs with nonsense or frameshift mutations within the three exons of this gene. Mutations in exons 1 or 2 reduce cytoplasmic mRNA accumulation whereas a mutation in exon 3 permits essentially normal expression. We report here that the post-transcriptional fate of mutated beta-globin mRNAs is differentially affected by the type of RNA polymerase II promoter driving expression. Replacement of the beta-globin promoter with the herpes simplex virus type 1 thymidine kinase gene promoter but not the cytomegalovirus immediate early promoter rescues the cytoplasmic accumulation of mutated mRNA to wild-type levels. This effect is shown to be independent of the absolute quantity and the kinetics of accumulation of mutated mRNA synthesized, and primer-extension analyses confirm that both viral promoters accurately utilize identical transcription start sites. These data thus reveal an unexpected property of RNA polymerase II promoters: determination of the post-transcriptional fate of the maturing mRNA, presumably by influencing alternative choices between as yet undefined processing and/or transport pathways.