Portion Of Gag Research Articles

Occupation of a thermoresistant-scaffold (αRep) at SP1-NC cleavage site disturbs the function of HIV-1 protease.

HIV-1 nucleocapsid (NC) becomes an attractive target for the development of novel anti-HIV-1 agents. Discovering of non-antibody scaffolds that disrupt the function of NC will be a potential aspect for disturbing viral maturation process. Correspondingly, we explored the specific binding site of the thermoresistant-scaffold protein, αRep9A8 which formerly demonstrated the inhibitory effect on HIV-1 replication. The portion of Gag, CA21-SP1-NC has been used as a template for designing nine overlapping peptides (P4–P12). The P9 peptide showed the strongest binding activity followed by P8 and P12 respectively. The amino acid sequences on those peptides resemble the N-terminal domain of the NC proximity to the SP1-NC initial cleavage site and across the conserved CCHC zinc finger 1 (ZF1) of NC. The interaction KD between αRep9A8 with its target was 224.9 ± 57.4 nM. Consequently, αRep9A8 demonstrated the interference of the HIV-1 protease function by hindering a protease cleavage site. The released NC product from CA21-SP1-NC was diminished. The present study provided an additional information of αRep9A8 function in interfering of viral maturation processes resulting in the decremental efficiency of viral infectivity.

Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells

The mouse mammary tumor virus (MMTV) Pr77Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77Gag-His6-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77Gag-His6-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His6-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77Gag-His6-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77Gag should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.

Patterns of genomic site inheritance in HIV-1M inter-subtype recombinants delineate the most likely genomic sites of subtype-specific adaptation.

Recombination between different HIV-1 group M (HIV-1M) subtypes is a major contributor to the ongoing genetic diversification of HIV-1M. However, it remains unclear whether the different genome regions of recombinants are randomly inherited from the different subtypes. To elucidate this, we analysed the distribution within 82 circulating and 201 unique recombinant forms (CRFs/URFs), of genome fragments derived from HIV-1M Subtypes A, B, C, D, F, and G and CRF01_AE. We found that viruses belonging to the analysed HIV-1M subtypes and CRF01_AE contributed certain genome fragments more frequently during recombination than other fragments. Furthermore, we identified statistically significant hot-spots of Subtype A sequence inheritance in genomic regions encoding portions of Gag and Nef, Subtype B in Pol, Tat and Env, Subtype C in Vif, Subtype D in Pol and Env, Subtype F in Gag, Subtype G in Vpu-Env and Nef, and CRF01_AE inheritance in Vpu and Env. The apparent non-randomness in the frequencies with which different subtypes have contributed specific genome regions to known HIV-1M recombinants is consistent with selection strongly impacting the survival of inter-subtype recombinants. We propose that hotspots of genomic region inheritance are likely to demarcate the locations of subtype-specific adaptive genetic variations.

Epigenetic analysis of HIV-1 proviral genomes from infected individuals: Predominance of unmethylated CpG's

Efforts to cure HIV-1 infections aim at eliminating proviral DNA. Integrated DNA from various viruses often becomes methylated de novo and transcriptionally inactivated. We therefore investigated CpG methylation profiles of 55 of 94 CpG's (58.5%) in HIV-1 proviral genomes including ten CpG's in each LTR and additional CpG's in portions of gag, env, nef, rev, and tat genes. We analyzed 33 DNA samples from PBMC's of 23 subjects representing a broad spectrum of HIV-1 disease. In 22 of 23 HIV-1-infected individuals, there were only unmethylated CpG's regardless of infection status. In one long term nonprogressor, however, methylation of proviral DNA varied between 0 and 75% over an 11-year period although the CD4+ counts remained stable. Hence levels of proviral DNA methylation can fluctuate. The preponderance of unmethylated CpG's suggests that proviral methylation is not a major factor in regulating HIV-1 proviral activity in PBMC's. Unmethylated CpG's may play a role in HIV-1 immunopathogenesis.

Growth ability in simian cells of monkey cell-tropic HIV-1 is greatly affected by downstream region of the vif gene

To obtain monkey-tropic (mt) HIV-1 derivatives with distinct biological characteristics and to improve the viral growth property, we have generated several variants from a prototype mt HIV-1 designated NL-DT5R (X4-tropic). The prototype HIV-1 contains a portion of gag and entire vif genes from SIVmac in its genome. The two derivatives carrying 3' half-genomic region of the SF162 (R5-tropic) or 89.6 (dual-tropic) isolate displayed very retarded or no viral growth, respectively, in a simian cell line HSC-F. In contrast, the three clones containing a part of env gene (encoding the V1-V4 region) from SF162, YU-2 (R5-tropic) or 89.6 showed different growth kinetics in HSC-F cells, although they grew somewhat more poorly than the NL-DT5R. Comparison of various viral proteins potentially involved in the different biological properties has revealed that, while amino acid sequences of Tat, Rev, Vpr, Vpu and Nef are quite conserved among the clones, those in the surface (SU) region of Env are relatively heterologous. Our data described here have shown that the 3' half of viral genome other than gag and vif genes greatly affects the growth property of mt HIV-1 in simian cells.

9. Mobilization of Sendai Virus Vectors (SeVV) by Phylogenetic Related Human Pathogens: Implications for Future Vector Generations

In an attempt to make HIV-1 based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of the TATA box and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this 400bp U3 deletion are thought to have no LTR-directed transcription, and are thus called self-inactivating (SIN) lentivectors. As with wild-type LTR vectors, any transcripts initiated at the U3-R junction in the 5' LTR of SIN lentivectors contain the packaging signal found in the psi region and 5' portion of gag. Transcripts containing the packaging signal have the potential to be encapsidated and mobilized by wild-type HIV-1. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5' LTR which is responsible for the production of full-length vector genome transcripts. To verify that transcripts derived from the LTR in SIN lentivectors are competent for encapsidation and integration, we transduced a lentiviral packaging cell line with a standard SIN lentivector and then observed production of viable vector particles containing full-length SIN lentivector genomes. We also demonstrate that a Herpes Simplex Virus thymidine kinase gene placed downstream of the SIN LTR, but upstream of the vector's internal promoter, is produced in quantities high enough to render cells containing this vector partially succeptible to gancyclovir-induced cell death. Additionally, we show that SIN lentivectors containing no internal promoter express an eGFP transgene. Using RT-PCR we demonstrate that transcripts initiated at the 5' LTR in SIN lentivectors can be readily detected, and using this assay, we conclude that the SIN LTR retains approximately 15% of the transcriptional activity of the wild-type LTR (in the absence of Tat). We have also identified sequences in the SIN lentivector which are responsible for transcriptional activation at the 5' LTR. With a promoter assay using different segments of the LTR and leader region of the SIN lentivector, we have determined that the residual promoter activity does not depend on any LTR sequence, including the HIV-1 start-site initiator (Inr) element. Rather, the promoter activity is contained entirely within the leader region, and although this element is downstream of the transcription initiation site, it is capable of initiating transcription from the 5' end of R in the LTR. Mutation of leader region binding sites for the transcriptional activators DBF1 and SP1 reduces transcription from the SIN LTR by up to 80%; however, a significant decrease in vector titer results from this mutation, perhaps owing to disruption of the folding of psi, which overlaps the SP1 binding site. In conclusion, knowledge of the potential for mobilization of SIN lentivectors by HIV-1 will be important for the design of future gene therapy trials using such vectors. It is also possible that low level expression from the SIN LTR may, in some instances, be adequate for the production of small amounts of transgene in a non-cell-type specific manner, which may be relevant to studies using SIN lentivectors to introduce cell-specific expression cassettes.

The human immunodeficiency virus type 1 carboxyl-terminal third of capsid sequence in Gag-Pol is essential but not sufficient for efficient incorporation of Pr160(gag-pol) into virus particles.

To elucidate the role of the C-terminal portion of Gag in the incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virus particles, a series of HIV-1 Gag-Pol mutants with deletions in the C-terminal gag sequence was constructed and viral incorporation of the Gag-Pol deletion mutants was analyzed using co-transfecting 293T cells with a Pr55(gag) expression plasmid. The biological function of the incorporated HIV-1 pol gene product was tested using an infectivity assay of the released virus particles which were pseudotyped with the murine leukemia virus Env. Analysis indicated that Gag-Pol deletion mutants, with a removal of the matrix (MA) and/or nucleocapsid (NC) or of the N-terminal two thirds of the gag coding sequence, could be incorporated efficiently into virus particles and produce significant amounts of infectious virions when assayed in a single-cycle infection assay. In contrast, mutations involving a deletion of the major homology region and the adjacent C-terminal capsid sequence significantly affected Gag-Pol incorporation. However, incorporation into virus particles of a Gag-Pol deletion mutant retaining both the major homology region and the adjacent C-terminal capsid intact was still severely impaired. This suggests that the capsid major homology region and the adjacent C-terminal capsid sequence in Gag-Pol are necessary but not sufficient for the incorporation of HIV-1 Pr160(gag-pol) into virus particles.

The Human Immunodeficiency Virus Type 1 Carboxyl-Terminal Third of Capsid Sequence in Gag-Pol Is Essential but Not Sufficient for Efficient Incorporation of Pr160gag-pol intoVirus Particles

To elucidate the role of the C-terminal portion of Gag in the incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virus particles, a series of HIV-1 Gag-Pol mutants with deletions in the C-terminal gag sequence was constructed and viral incorporation of the Gag-Pol deletion mutants was analyzed using cotransfecting 293T cells with a Pr55^gag expression plasmid. The biological function of the incorporated HIV-1 pol gene product was tested using an infectivity assay of the released virus particles which were pseudotyped with the murine leukemia virus Env. Analysis indicated that Gag-Pol deletion mutants, with a removal of the matrix (MA) and/or nucleocapsid (NC) or of the N-terminal two thirds of the gag coding sequence, could be incorporated efficiently into virus particles and produce significant amounts of infectious virions when assayed in a single-cycle infection assay. In contrast, mutations involving a deletion of the major homology region and the adjacent C-terminal capsid sequence significantly affected Gag-Pol incorporation. However, incorporation into virus particles of a Gag-Pol deletion mutant retaining both the major homology region and the adjacent C-terminal capsid intact was still severely impaired. This suggests that the capsid major homology region and the adjacent C-terminal capsid sequence in Gag-Pol are necessary but not sufficient for the incorporation of HIV-1 Pr160^gag-pol into virus particles.

Enhanced cellular immune responses to SIV Gag by immunization with influenza and vaccinia virus recombinants

An effective vaccination strategy against human immunodeficiency virus type 1 (HIV-1) should include the induction of potent cellular immune responses against conserved HIV-1 antigens. We have generated five replication competent recombinant influenza viruses (rFlu/SIV Gag nos. 1–5) expressing different portions of Gag of simian immunodeficiency virus (SIV). Single intranasal immunizations in mice with each rFlu/SIV Gag viruses resulted in different degrees of protection against a challenge with recombinant vaccinia virus expressing SIV Gag. Immunized BALB/c mice had detectable CD8+ T cell responses specific for Gag peptide 185–199 when mice were vaccinated with rFlu/SIV Gag no. 3 virus, and for Gag peptides 281–295 and 285–299 when vaccinated with rFlu/SIV Gag no. 4 virus. Cellular immune responses against SIV Gag were further enhanced by a booster with a recombinant vaccinia virus expressing SIV Gag in both the spleen and local lymph node tissues, resulting in the induction of robust Gag-specific CD8+ T cell responses at both systemic and mucosal levels. We suggest that a prime-boost immunization regimen using recombinant influenza and vaccinia viruses expressing HIV Gag might represent an effective means to induce potent HIV-specific, protective CD8+ T cell responses.

Characterization of Moloney murine leukemia virus p12 mutants blocked during early events of infection.

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3' ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.

Alphavirus replicon particles as candidate HIV vaccines.

Replicon particles based on Venezuelan equine encephalitis virus (VEE) contain a self-replicating RNA encoding the VEE replicase proteins and expressing a gene of interest in place of the viral structural protein genes. Structural proteins for packaging of replicon RNA into VEE replicon particles (VRPs) are expressed from separate helper RNAs. Aspects of the biology of VEE that are exploited in VRP vaccines include 1) expression of very high levels of immunogen, 2) expression of immunizing proteins in cells in the draining lymph node, and 3) the ability to induce mucosal immunity from a parental inoculation. Results of experiments with VRPs expressing green fluorescent protein or influenza virus hemagglutinin (HA) demonstrated that specific mutations in the VRP envelope glycoproteins affect both targeting in the draining lymph node and efficiency of the immune response in mice. VRPs expressing either the matrix-capsid portion of Gag, the full-length envelope gp160, or the secreted gp140 of cloned SIVsm H-4i were mixed in a cocktail and used to immunize macaques at 0, 1, and 4 months. Neutralizing antibodies against SIVsm H-4 were induced in 6 of 6 vaccinates and CTL in 4 of 6. An intrarectal challenge with the highly pathogenic SIVsm E660 was given at 5 months. A vaccine effect was seen in reduced peak virus loads, reduced virus loads both at set point and at 41 weeks postchallenge, and preserved or increased CD4 counts compared to controls. A candidate VRP HIV vaccine expressing Clade C Gag contains a sequence that is very close to the South African Clade C consensus and was selected from a recent seroconverter in the Durban cohort to represent currently circulating genotypes in South Africa. A GMP lot of this vaccine has been manufactured and tested for a phase I trial in the first months of 2002.

The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

Inhibition of human immunodeficiency virus type 1 by packageable, multigenic antisense RNA.

Viral-based vectors can provide an efficient delivery mechanism for stable expression of antisense RNA. To enhance and propagate the antiviral effect of antisense RNA, two novel human immunodeficiency virus type 1 (HIV-1)-based vector DNAs, designated as pMAG7 and pMAG19, were constructed which contained HIV-1 cis-acting packaging elements and produced multigenic HIV-1 antisense RNA that could target the entire pol, env, vif, vpu, vpr, rev, and tat and portions of gag and nef. The two DNAs were identical except that pMAG19 had additional gag coding sequences. Cotransfection of pMAG DNA and infectious, cloned HIV-1 DNA in 293 cells inhibited virus production (81%-98% reduction in reverse transcriptase activity) of various T cell-tropic and macrophage-tropic clade B isolates, such as NL4-3, YU-2, and JR-CSF. In addition, virion-associated pMAG antisense RNA was detected in residual virus particles produced by pNL4-3 in the presence of pMAG7 DNA, and the antisense sequences were stably transferred by infection of 174 x CEM cells. The results suggest that pMAG DNA may confer broad protection against HIV-1 by reducing initial virus burden due to antisense RNA and subsequent virus spread by propagation of antisense sequences along with wild-type virus.

The v-myc oncogene.

v-myc is the viral homolog of c-myc transduced by several acute transforming retroviruses, many of which encode this gene as a Gag-Myc fusion protein. The v-myc oncogene can transform several lineages of mammalian and avian cells either alone or in cooperation with other oncogenes. While the Gag portion of the Gag-Myc fusion protein and the nuclear localization signal each appear to be dispensable for transformation, the N- and C-termini of the Myc sequence have been found to be essential for transformation. All v-myc genes contain point mutations which seem to confer a greater potency to v-myc in the process of transformation, proliferation, and apoptosis. In v-myc-transformed myelomonocytic cells, secondary events occur, such as the expression of colony stimulating factor-1 (CSF-1) which play a critical role in immortalization and subsequent tumor progression. Inhibition of the autocrine loop of CSF-1 was found to induce apoptosis in the immortalized cells. While overexpression of v-Myc blocks terminal differentiation of hematopoietic cells, this is not sufficient to block the differentiation of certain neural and skeletal muscle cells. Recent developments on the effects of v-myc on cell growth, transformation, differentiation and apoptosis are discussed in this review.

NMR structure of the (52–96) C-terminal domain of the HIV-1 regulatory protein vpr: molecular insights into its biological functions

The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52–96 but not 12–51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52–96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30 %) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53–78) amphipathic α-helix, followed by a less defined (79–96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the α-helix still exists in (52–96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52–96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.

Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system.

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.

In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles.

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space. We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V. M. Vogt, J. Virol. 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA. A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size. By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone. Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro. We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.

Proteolytic Activity of Purified Avian Sarcoma and Leukemia Virus NC-PR Protein Expressed inEscherichia coli

Processing of the internal structural and enzymatic proteins of retroviruses occurs during or shortly after budding and is accomplished by the viral protease (PR), which belongs to the large family of aspartic proteases. It is not known how the activity of PR is regulated so that proteolysis occurs at this time. Cellular aspartic proteases are synthesized as zymogens with short N-terminal extensions that are proteolytically removed to generate the free active enzyme. In the avian sarcoma and leukosis viruses (ASLV), PR is expressed as the carboxy-terminal domain of the Gag polyprotein, which thus has a structure analogous to such a zymogen. We have investigated the enzymatic properties of ASLV PR when it is part of a longer protein, NC-PR, serving as a model for Gag. This protein represents about one-third of Gag and consists of the nucleocapsid (NC) domain fused to the N-terminus of PR. NC-PR and derivatives of NC-PR were expressed in bacterial cells and purified. In short-term assays, these fusion proteins lacked measurable protease activity toward an exogenous substrate prepared byin vitrotranslation. In contrast to PR, which is a homodimer, NC-PR migrated as a monomer both by glycerol gradient sedimentation and by gel filtration chromatography. Thus the NC domain appears to inhibit enzymatic activity by altering the dimerization potential of the PR domains. However, upon long incubations NC-PR was found to cleave itself to generate free and fully active PR, implying that dimerization was not prevented entirely. On the basis of these results, we hypothesize that the Gag proteinin vivois also incompletely active as a protease, because upstream portions of Gag interfere with proper interaction of the PR domains. The eventual dimerization, perhaps triggered by other events, then could lead to a cascade whereby PR is proteolytically freed from Gag and thereby gains enzymatic activity.

RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1.

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.

Characterization of the transforming domain of a feline sarcoma virus encoding a fgr-related tyrosine kinase.

We investigated the transforming domain of a recently isolated feline sarcoma virus (TP1-FeSV) which encodes a fgr-related tyrosine kinase expressed as a gag-fgr fusion protein. The gag portion was removed and replication-competent expression vectors (RCAS) with inserted v-fgr sequences were established. Chicken embryo fibroblasts (CEF) were transfected and monitored for replication, integration and transcription of the proviral constructs. We demonstrated that transfected cells display morphological changes and are able to form colonies in soft-agar. This suggests that the gag portion of the fusion protein from TP1-FeSV is not necessary for the transformation of fibroblasts.