Targeting of murine leukemia virus gag to the plasma membrane is mediated by PI(4,5)P2/PS and a polybasic region in the matrix.

Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P(2) only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P(2) depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P(2) appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P(2) binding cleft.

SIVMAC Vpx improves the transduction of dendritic cells with nonintegrative HIV-1-derived vectors.

Lentiviral vector (LV)-mediated gene therapy bears an intrinsic risk of insertional mutagenesis following integration into the host genome. Nonintegrative LVs may offer an alternative avenue at least in nondividing cells where episomal viral DNA persists stably. Owing to their central role in immune system functions, differentiated dendritic cells (DCs) offer an interesting cell target for these vectors. We have previously described that the transduction of DCs with wild-type HIV-1-derived vectors can be considerably improved by providing DCs with noninfectious virion-like particles (VLPs) carrying Vpx (Vpx-VLPs), a nonstructural protein coded by members of the SIV(SM)/HIV-2 lineage that removes a specific restriction to lentiviral infection in these cells. Here, we describe that the transduction efficiency of DCs with nonintegrative HIV-1 vectors can also be improved via Vpx-VLPs that promote the accumulation of complete and episomal viral DNA. In this setting, Vpx increases both the number of transduced cells and the levels of transgene expression. Thus, these results describe a simple procedure by which transduction of differentiated DCs can be achieved at low viral inputs with safer LVs to improve both the number of transduced cells and the levels of transgene expression.

Control of Rous sarcoma virus RNA translation and packaging by the 5' and 3' untranslated sequences.

A cytopathic mutant of Rous sarcoma virus-PrB was isolated and shown to have two large deletions, which result in the junction of Gag sequences in P27 to the 5' end of Env, and in the loss of the Src gene. This replication-defective (rd) and transformation-defective (td) mutant can replicate in the presence of its helper, which is also td, but the viral particles produced are poorly infectious. Most of the virions do not contain viral RNA, and the mutant RNA accumulates in infected cells, where it is poorly translated and packaged. Molecular clones of the mutant, of its helper and of a PrBtd strain were obtained in lambda-EMBL3, characterized, shown to be biologically active by transfection assays and sequenced. Nucleotide sequence comparisons indicate that the strong ribosome-binding site of Rous sarcoma virus RNA, responsible for the efficient RNA translation in vivo and in vitro, is mutated in PrB-(HM) mutant RNA; this causes the inhibition of RNA translation, as demonstrated by translation competition experiments using virus RNA made in vitro that carries the original or the mutated ribosome-binding site. In addition, an insertion present at the 3' end of both the mutant and the helper RNA, but absent in PrBtd RNA, is probably responsible for the inhibition of RNA packaging. Finally, these data are discussed in the light of a model of a 5'----3' Rous sarcoma virus RNA structure leading to a circular RNA molecule, which has implications in RNA translation, packaging and reverse transcription.

Possible roles of nucleocapsid protein of MoMuLV in the specificity of proviral DNA synthesis and in the genetic variability of the virus.

Retroviral nucleocapsid (NC) protein, in addition to its structural roles in the virion core, is involved in the early and late phases of the viral replication cycle. To further characterise the role of NC protein of MoMuLV (NCp10) in the replication of the viral genome, the influence of NCp10 on self-primed versus primer-specific reverse transcription has been analysed in vitro. The results show that NCp10 can enhance the specificity of proviral DNA synthesis by inhibiting self-primed cDNA synthesis while promoting primer-specific DNA synthesis within active NCp10-RNA nucleoprotein complexes. Retroviruses are known to show a high degree of variability and this prompted us to examine the possible implication of NCp10 in the genetic variability of MoMuLV. The ability of reverse transcriptase (RT) to extend different mutated primers using an RNA or a DNA template has been investigated in the presence or in the absence of NCp10. NCp10 was found to have different effects on RT depending on the nature of the template: an enhancement at the elongation level of mutated primers using RNA as template versus a slight inhibition using DNA as template. These observations suggest that NCp10 could be implicated in the genetic variability of MoMuLV by allowing nucleotide misincorporation principally during minus strand DNA synthesis.

It is Rous sarcoma virus protein P12 and not P19 that binds tightly to Rous sarcoma virus RNA.

The interactions between Rous Sarcoma virus (RSV) RNA and the viral proteins in the virus have been analysed by Sen & Todaro (1977) using ultraviolet light irradiation; they showed that the major protein ultraviolet light cross-linked to the viral RNA was P19 as identified by polyacrylamide gel electrophoresis. We report here that it is not viral protein P19 but P12 that binds tightly to RSV RNA upon ultraviolet light irradiation of the virus. Therefore, the binding sites of the viral protein along RSV RNA that we have characterized previously should be correctly attributed now to P12 and not P19.

Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus.

A sequence of the rat retrotransposon virus-like 30 S RNA (VL30) located next to the 5' end of the Harvey murine sarcoma virus (HaMSV) genome was recently found to form stable dimeric RNA in vitro and to direct the efficient packaging of VL30-derived recombinant RNAs into MuLV virions. To study the structure-function relationships of the rat VL30 dimerization-encapsidation signal (E/DLS), we have performed biochemical and genetic studies of rat VL30 RNA dimerization in vitro. The results show that temperature and specific cation/RNA interactions are important for VL30 dimerization in vitro. VL30 RNA dimerization is optimal at 55 degrees C and Li+ dramatically enhances the stability of VL30 dimeric RNA. In addition, a genetic analysis of VL30 RNA dimerization reveals that a 5' G-rich sequence is critical for dimer formation and that a UGUCUUGUC repeat contributes to VL30 dimer stability. Interestingly enough, substitution of an A for a G in the 5' G-rich sequence is sufficient to abolish VL30 RNA dimerization in vitro. Taken together, these biochemical and genetic data indicate that dimerization of VL30 RNA involves non-canonical base-pairings and possible purine-purine interactions. Nucleocapsid protein NCp10 of murine leukemia virus (MuLV), a gag-encoded protein that is tightly associated with genomic RNA in the virion core, has been shown to have nucleic acid binding and annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to the retrotransposon VL30 RNA and to activate its dimerization. Moreover, mutations in the 5' G-rich sequence of the VL30 dimerization sequence impaired NCp10 binding to RNA. Recombinant MLV-VL30 vectors with mutations in the VL30 dimerization sequence were constructed. Results obtained in vivo clearly show that the mutations that had a deleterious effect on the packaging of MLV-VL30 retroviral vector in vivo were those that impaired VL30 RNA dimerization and interactions with NCp10 in vitro, even the single mutation in the 5' G-rich region. Therefore, these findings suggest that packaging of VL30 RNA into MuLV virions requires specific interactions between RNA dimerization sequences and viral NC protein molecules.

CUG initiation codon used for the synthesis of a cell surface antigen coded by the murine leukemia virus.

Murine leukemia virus (MuLV) codes for two precursors of the group-specific antigens, Pr65gag and Pr75gag, in vivo. While Pr65gag is the precursor to the virion structural proteins, Pr75gag undergoes glycosylation and is found on the surface of the infected cell as gp85gag, and it is thought to play a role in virus maturation and spread. Pr65gag synthesis starts at an AUG codon within a favourable initiation context (AAUAUGG at positions 618 to 624). The gp85gag start codon is upstream but its precise location is not known. To map the initiation codon of gp85gag, we used deletion and site-directed mutagenesis of the leader sequence of MuLV RNA and in vitro translation of the RNAs. Synthesis of the MuLV gp85gag protein appears to be initiated at a CUG codon located within a favourable context (ACCCUGG at positions 354 to 359 for Moloney-MuLV). The possible function of gp85gag was investigated by expressing Moloney-MuLV and Friend-MuLV proviral DNA and mutants deficient for gp85gag synthesis in mouse and rat cells. The results indicate that the gp85gag protein probably facilitates the spread of virus infection in tissue culture.

Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase.

Packaging of the genomic RNA dimer and replication primer tRNA(Lys,3) into HIV virions are required for the production of infectious virus. The initiation of reverse transcription necessitates the annealing of tRNA(Lys,3) to the primer binding site (PBS) of HIV RNA by nucleocapsid (NC) protein. In this report the interactions of replication primer tRNA(Lys,3) with various forms of reverse transcriptase (RT) and nucleocapsid protein have been analyzed by ultraviolet light (UV) cross-linking and gel retardation assays. We show that of the three forms of RT studied, p66/p51, p66 and p51, only the heterodimer p66/p51 can tightly and stably interact with tRNA(Lys,3). Tight interactions between tRNA(Lys,3) and nucleocapsid protein, either NCp15 or NCp7, were found to take place within the anticodon domain. Interestingly enough, primer tRNA(Lys,3) can interact with RTp66/p51 and NCp15 to form a high molecular weight complex in which RTp66/p51 appears to enhance the binding of NCp15 to tRNA(Lys,3). These findings favor the notion that the RT enzyme and NC protein co-operate to select and package primer tRNA.

Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.

Characterization of loose and tight dimer forms of avian leukosis virus RNA.

Retroviral genomes consist of two identical RNA molecules joined non-covalently near their 5'-ends. Recently, we showed that an imperfect autocomplementary sequence, located in the L3 domain, plays an essential role in avian sarcoma-leukosis virus (ASLV) RNA dimerization in vitro. This sequence can adopt a stem-loop structure and is involved in ASLV replication. Here, we found that in the absence of nucleocapsid protein, RNA transcripts of avian leukosis virus (ALV) were able to form two types of dimers in vitro that differ in their stability: a loose dimer, formed at a physiological temperature, and a tight dimer, formed at a high temperature. A mutational analysis was performed to define the features of these dimers. The results of this analysis unambiguously confirm that the two L3 stem-loops interact directly in both types of dimers. A loop-loop interaction is the main linkage in the loose dimer. In contrast, in the tight dimer, the stem and the loop of the L3 hairpin form an extended duplex. Surprisingly, we also found that the dimerization properties defined for our ALV strain (type SR-A) differ from those found in other ASLV strains.

Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA.

In Moloney murine leukemia virus, the encapsidation Psi element was shown to be necessary and sufficient to promote packaging of viral RNA, and to be required for dimerization. The conformation of the Psi domain (nucleotides 215 to 565) was investigated in solution by chemical probing. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate at cytosine N3 and adenosine N1, and with a carbodiimide derivative at guanosine N1 and uridine N3. Position N7 of adenine residues was probed with diethylpyrocarbonate. The analyses were conducted on in vitro transcribed fragments corresponding either to the isolated Psi domain or to the 5'-terminal 725 nucleotides. The RNA fragments were analyzed in their monomeric and dimeric forms. A secondary structure model was derived from probing data, computer prediction and sequence analysis of related murine retroviruses. One major result is that Psi forms an independent and highly structured domain. Dimerization induces an extensive reduction of reactivity in region 278 to 309 that can be interpreted as the result of intermolecular interactions and/or intramolecular conformational rearrangements. A second region (around position 215) was shown to display discrete reactivity changes upon dimerization. These two regions represent likely elements of dimerization. More unexpectedly, reactivity changes (essentially enhancement of reactivity) were also detected in another part of Psi (around position 480) not believed to contain elements of dimerization. These reactivity changes could be interpreted as dimerization-induced allosteric transitions.

First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses.

Retroviruses are a family of widespread small animal viruses about 110 nm in diameter, composed of an inner core surrounded by an outer envelope formed of a lipid bilayer of cellular origin in which are anchored viral glycoproteins. The inner core is formed by an outer shell of capsid protein molecules (CA protein) surrounding the dimeric RNA genome in close association with about 2000 molecules of nucleocapsid protein (NC protein) and molecules of reverse transcriptase (RT) and integrase (IN). Conversion of the genomic single-stranded RNA into a double-stranded proviral DNA by RT takes place in the nucleocapsid substructure and involves two DNA strand transfers to generate the long terminal repeats (LTR) required for IN-mediated integration of the proviral DNA into the cellular genome and its expression. In this review we have summarized some of the properties and functions of the nucleocapsid protein of the most intensely studied oncoretroviruses (MuLV and ASLV) and lentiviruses (HIV-1). Recent biochemical and genetic data on retroviral NC proteins have shown that this small viral protein endowed with a strong affinity for nucleic acids exhibits nucleic acid annealing and strand transfer activities and is required for the formation of infectious viral particles. These new activities of NC protein are most probably necessary at the early steps of proviral DNA synthesis. The 3-D structures of HIV-1 and MoMuLV NC proteins, deduced from NMR studies, are characterized by a central globular domain with one (MoMuLV) or two (HIV-1) zinc fingers. This should facilitate a rational approach of new anti-HIV therapies based on inhibition of NC protein functions. Due to space limitations and the very abundant literature on retroviruses, references to articles prior to the publication of the second volume of RNA Tumor Viruses in 1985 (Weiss et al., 1985) will be minimal. We also direct the reader to an excellent review which summarizes recent insights into biochemical and structural aspects of the retroviral enzymes PR, RT and IN (Katz & Skalka, 1994).

Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains.

The 5' region of HIV-1 RNA contains functional elements involved in key steps of the retroviral cycle, such as genomic RNA transcription, splicing, translation, dimerization or initiation of reverse transcription. In the present work, we investigated the conformation of the first 500 nucleotides covering the RNA leader and the 5' gag coding sequences of HIV-MAL, using chemical probing. We provide detailed information on almost each nucleotide at one of their Watson-Crick positions and on position N-7 of purines. Experiments were conducted on two in vitro transcribed RNA fragments (1 to 707 and 1 to 311). A secondary structure model was derived by combining the experimental data, computer predictions and sequence comparison. Under conditions favoring dimerization (high salt concentration), HIV-1 RNA folds into independent structural domains that can be related to defined functional regions. The first domain corresponds to TAR forming a stable stem-loop. Intrinsic structural features are found to stabilize the TAR hairpin loop. The second domain (nucleotides 56 to 299) contains the PBS sequence, which is located in a stable subdomain constrained by a four stem junction (nucleotides 139 to 218). Although the MAL isolate has an insertion near the PBS, probably resulting from the duplication of a 23-nucleotide sequence, the structural organization of this subdomain is conserved in all other HIV-1 isolates. The third domain (nucleotides 300 to 404) contains the splice donor site, packaging and dimerization elements and the AUG initiation codon of gag. A major result is the structural versatility of this region. Two mutually exclusive structures, both equally in agreement with probing data, could modulate the different functions involving this domain. The reduced accessibility of the gag translational initiation site possibly accounts for the low efficiency of the in vitro translation of the dimer. Finally, the 5' gag coding sequences form a metastable domain.

CIS elements and trans-acting factors required for minus strand DNA transfer during reverse transcription of the genomic RNA of murine leukemia virus.

During reverse transcription of the retroviral genomic RNA, two obligatory DNA strand transfers take place to synthesize the complete proviral DNA with two LTRs. We have previously shown that using an in vitro system made up of two viral RNAs mimicking the 5' and 3' regions of the retroviral genome, both nucleocapsid protein and the repeat (R) sequences were necessary for minus strong-stop cDNA (ss-cDNA) transfer and elongation by reverse transcriptase (RT). In this paper we show that the basic residues of nucleocapsid protein NCp10 of Moloney murine leukemia virus (MoMuLV), but not the zinc finger, are necessary for minus strand transfer. In order to examine the role of the R sequence repeated at the 5' and 3' ends of the genome in minus strand DNA transfer, the MoMuLV R sequence of 68 nt was replaced by either HIV-1 R of 96 nt, or RSV R of 21 nt, or by an artificial sequence of 21 nt. Analysis of MoMuLV DNA strand transfer from the 5' RNA to the 3' RNA and elongation in the presence of NCp10 and RT showed that it was high with control MoMuLV R, high with RSV R, reduced with HIV-1 R, and undetectable with the artificial R sequence. These results suggest that minus strand DNA transfer is a process more complex than simple hybridization of ss-cDNA to the 3' R sequence of the genomic RNA.

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

Conformational behaviour of the active and inactive forms of the nucleocapsid NCp7 of HIV-1 studied by 1H NMR.

The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo.

Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors.

The murine leukemia virus (MLV)-related type C viruses constitute a major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5' untranslated leader involved in key steps of the viral life cycle--splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5' leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

Stable MLV-VL30 dicistronic retroviral vectors with a VL30 or MoMLV sequence promoting both packaging of genomic RNA and expression of the 3' cistron.

The Friend murine leukemia virus (MLV) and VL30 5' leader sequences were recently found to possess an internal ribosomal entry signal (IRES) and promote cap-independent translation of a downstream cistron. The use of an IRES to generate a dicistronic message provides a way to express two exogenous proteins efficiently and stably within cells. In the present study, we used the VL30 and Moloney (Mo)MLV 5' leader sequences with both RNA translation (IRES) and packaging (E/DLS) functions to construct dicistronic retroviral vectors designed to express human placental alkaline phosphatase (plap) and neomycin phosphotransferase (neo). Vectors containing the VL30 (positions 205-794) and MoMLV (positions 210-1,035) 5' sequences between two cistrons were able to direct synthesis of exogenous proteins and were produced at a high titer, indicating that the IRES and packaging elements were functional in such dicistronic retroviral vectors. In addition, long-term selection for neo expression did not impair plap gene activity. In general, no major genetic rearrangement of the integrated recombinant provirus was observed with the dicistronic constructs upon prolonged culture of the infected cells.