Rev-free HIV-1 gene delivery system for targeting Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway

The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T-cells. Here, we investigated the use of RNA transport elements from Mason-Pfizer monkey virus or MPMV for the creation of high-titered Rev-free HIV-1-based packaging systems. The HIV-1 gag/pol expression constructs containing one or more copies of MPMV constitutive RNA transport element (CTE) were used to package similarly modified gene-transfer vectors in the presence or absence of Rev. An inverse correlation between the number of CTE modules and Rev dependency was noted for vector stock production. While packaging systems containing multiple CTEs were resistant to exogenously expressed Rev M10, the titers of vectors encoding Rev M10 were nevertheless reduced in comparison to vectors encoding only green fluorescent protein (GFP). In contrast, a gene transfer vector encoding the Rev M10 transgene and containing both RNA transport elements exhibited almost no loss in titer in comparison to a corresponding vector encoding only GFP. The optimized Rev-independent gene delivery system was used for delivery of Rev M10 transgene into T-lymphocytes. Upon challenge in single round infection assays with HIV-1, the modified T-cells produced fewer virus particles than control cells expressing GFP. This Rev-free packaging system may prove useful for targeting the Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway for inhibiting HIV replication.     

Co-packaging of sense and antisense RNAs: a novel strategy for blocking HIV-1 replication.

Retroviral vectors were engineered to express either sense (MoTiN-TRPsie+) or sense and antisense (MoTN-TRPsie+/-) RNAs containing the human immunodeficiency virus type-1 (HIV-1) trans -activation response (TAR) element and the extended packaging (Psie) signal. The Psie signal includes the dimer linkage structure (DLS) and the Rev response element (RRE). Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid (MT4) cell line. Stable transductants were then tested for sense and antisense RNA production and susceptibility to HIV-1 infection. HIV-1 production was significantly decreased in cells transduced with MoTiN-TRPsie+ and MoTN-TRPsie+/-vectors. Efficient packaging of sense and most remarkably of antisense RNA was observed within the virus progeny. Infectivity of this virus was significantly decreased in both cases, suggesting that the interfering RNAs were co-packaged with HIV-1 RNA. Vector transduction was not expected to occur and was not observed. Inhibition of HIV-1 replication was also demonstrated in human peripheral blood lymphocytes transduced with retroviral vectors expressing antisense RNA. These results suggest that (i) both sense and antisense RNAs were co-packaged with HIV-1 RNA, (ii) the co-packaged sense and antisense RNAs inhibited virus infectivity and (iii) the co-packaged sense and antisense RNAs were not transduced. Sense and antisense RNA-based strategies may also be used to co-package other interfering RNAs (e.g. ribozymes) to cleave HIV-1 virion RNA.     

HIV-1 Suppressive Sequences Are Modulated by Rev Transport of Unspliced RNA and Are Required for Efficient HIV-1 Production

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3′ untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3′ untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.     

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.     

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.     

Continuous high-titer HIV-1 vector production

Human immunodeficiency virus type 1 (HIV-1)-based vectors are currently made by transient transfection, or using packaging cell lines in which expression of HIV-1 Gag and Pol proteins is induced. Continuous vector production by cells in which HIV-1 Gag-Pol is stably expressed would allow rapid and reproducible generation of large vector batches. However, attempts to make stable HIV-1 packaging cells by transfection of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only low levels of p24 antigen (20-80 ng/ml), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. Here we report that expression of HIV-1 Gag-Pol by a murine leukemia virus (MLV) vector allows constitutive, long-term, high-level (up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were constructed using codon-optimized HIV-1 Gag-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 10(7) 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)     

HIV-1 Vpr potently induces programmed cell death in the CNS in vivo

The human immunodeficiency virus type I (HIV-1) accessory protein Vpr has been associated with the induction of programmed cell death (apoptosis) and cell-cycle arrest. Studies have shown the apoptotic effect of Vpr on primary and established cell lines and on diverse tissues including the central nervous system (CNS) in vitro. However, the relevance of the effect of Vpr observed in vitro to HIV-1 neuropathogenesis in vivo, remains unknown. Due to the narrow host range of HIV-1 infection, no animal model is currently available. This has prompted us to consider a small animal model to evaluate the effects of Vpr on CNS in vivo through surrogate viruses expressing HIV-1Vpr. A single round of replication competent viral vectors, expressing Vpr, were used to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo. Viral particles pseudotyped with VSV-G or N2c envelopes were generated from spleen necrosis virus (SNV) and HIV-1-based vectors to transduce CNS cells. The in vitro studies have demonstrated that Vpr generated by SNV vectors had less apoptotic effects on CNS cells compared with Vpr expressed by HIV-1 vectors. The in vivo study has suggested that viral particles, expressing Vpr generated by HIV-1-based vectors, when delivered through the ventricle, caused loss of neurons and dendritic processes in the cortical region. The apoptotic effect was extended beyond the cortical region and affected the hippocampus neurons, the lining of the choroids plexus, and the cerebellum. However, the effect of Vpr, when delivered through the cortex, showed neuronal damage only around the site of injection. Interestingly, the number of apoptotic neurons were significantly higher with HIV-1 vectors expressing Vpr than by the SNV vectors. This may be due to the differences in the proteins expressed by these viral vectors. These results suggest that Vpr induces apoptosis in CNS cells in vitro and in vivo. To our knowledge, this is the first study to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo in neonatal mice. We propose that this, in expensive animal model, may be of value to design-targeted neuroprotective therapeutics.     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

Incorporation of Pr160(gag-pol) into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag-Pol polyprotein.

The determinants critical for the incorporation of Pr160(gag-pol) into human immunodeficiency virus type 1 (HIV-1) particles were examined by cotransfecting cells with (i) a plasmid expressing wild-type Gag protein and (ii) a series of chimeric Gag-Pol expression plasmids in which individual murine leukemia virus (MLV) Gag regions and subdomains precisely replaced their HIV-1 counterparts. The presence of the MLV MA and NC Gag regions in the chimeric Gag-Pol precursor had no detectable effect on the incorporation of Gag-Pol into progeny virions. In contrast, the entire HIV-1 CA region was required to achieve wild-type levels of Gag-Pol assembly into particles; both the CA major homology region and the adjacent C-terminal CA sequences play dominant roles in this process yet, when assayed in the context of a chimeric Gag-Pol polyprotein, restored the defect affecting Gag-Pol incorporation to approximately half of the wild-type level.     

Overexpression and incorporation of GagPol precursor does not impede packaging of HIV-1 tRNA<Superscript><Emphasis Type="Italic">Lys3</Emphasis></Superscript> but promotes intracellular budding of virus-like particles

We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/Gag-Pol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNA ^Lys3 . In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNA ^Lys3 . Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(-)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(–) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(–) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(–) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.     

Selection and Characterization of Human Immunodeficiency Virus Type 1 Mutants That Are Resistant to Inhibition by the Transdominant Negative RevM10 Protein

Intracellular immunization with RevM10, a transdominant negative form of the Rev protein, efficiently inhibits human immunodeficiency virus (HIV) replication in vitro and gene therapy protocols that use this modality are currently being evaluated in human clinical trials. Development of resistance to this kind of therapy has not been previously reported. Here we show that RevM10-resistant HIV type 1 (HIV-1) variants can be selected by in vitro passage of HIV-1 in a T-lymphoblastoid cell line constitutively expressing RevM10. Unexpectedly, the selected variants showed changes in the Rev response element (RRE) but no changes in Rev. Replacement of the wild-type RRE with a mutated RRE resulted in a virus that showed increased resistance to RevM10. After repeated passages of the resistant variant in cells expressing RevM10, a virus with an additional mutation in the viral vpu gene was selected. Surprisingly, a virus containing only this vpu mutation also showed some resistance to inhibition by RevM10.     

Multiple restrictions of human immunodeficiency virus type 1 in feline cells

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.