Lentiviruses can infect mitotic and non-dividing cells owing to the karyophilic properties of their pre-integrating complex, which allow its active import through the nucleopore. Thus lentiviral vectors derived from human immunodeficiency virus type 1 can mediate an efficient transfer integration, and stable expression of transgenes into proliferating and stationary cells both in vivo and in vitro. By adopting the internal ribosome entry site of encephalomyocarditis virus for bicistronic expression or two promoters of EF-1alpha and SV40 for separate expression of two genes of interest, we developed two lentiviral vectors that express two genes. On FACS analysis, RT-PCR, and immunofluorescence assay, it was shown that the target cells expressed two genes of interest at different levels as the transducing vectors designed for. This vector system is useful especially for a stable, dual-gene expression and two transgene deliveries to non-dividing cells.
Encephalomyocarditis virus ; Fluorescent Antibody Technique ; HIV-1 ; Lentivirus ; Peptide Elongation Factor 1 ; Ribosomes ; Transgenes*

OBJECTIVETo develop a safe and effective lentivirus vaccine model and provide insights into the development of other lentivirus vaccines.

METHODSIn this study, a construct of pGPT was made by deleting env gene in the infectious Equine infectious anemia virus (EIAV) molecular clone of WU57. Since the overlaping of EIAV Rev gene with env gene, there was no Rev gene in the construct of pGPT. For compensation of Rev function, the construct of pGPTC was made by inserting 4 copies of constitutive RNA transport elements (CTEs) from Mason-Pfizer monkey virus into the construct of pGPT. In addition, a construct designated pTEB expressing EIAV Env protein was made while env gene-minus viruses were made by co-transfection of pGPT/pTEB or pGPTC/pTEB into 293 cells. Western blot was used to identify the development of recombinant virus particles. Then immunofluorescence assay was used to evaluate the infectivity of recombinant virus particles in vitro.

RESULTSEIAV proteins expression was detected in the supernatant of transfected 293 cells by Western blot within pGPTC/pTEB transfected cells. However, no evidence of EIAV proteins expression was observed within pGPT/pTEB transfected cells. EIAV proteins expression was detected in the first round but not in the second round infected EK cells with EIAV(GPTC) by immunofluorescence assay.

CONCLUSIONRev/RRE was necessary for expression of viral structural proteins; CTEs from Mason-Pfizer monkey virus was functionally interchangeable with EIAV Rev/RRE to help RNAs transportation out of nucleus to express structural proteins and EIAV particles were produced in the transfected 293 cells. A live EIAV recombinant virus with single round infection had been developed.

Animals ; Blotting, Western ; Cells, Cultured ; Fluorescent Antibody Technique ; Genes, rev ; Haplorhini ; Horses ; Humans ; Infectious Anemia Virus, Equine ; genetics ; Lentivirus ; immunology ; Lentivirus Infections ; immunology ; prevention & control ; Mason-Pfizer monkey virus ; genetics ; Transfection ; Vaccines, Synthetic ; Viral Vaccines ; genetics ; immunology

We previously demonstrated that the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 gp41 induced cell death in human neuronal cells. Present study was conducted to further elucidate the pathogenic mechanisms involved in HIV-1 gp41-induced neurodegeneration in AIDS patients with cognitive deficits. The effect of LLP-1 on activation of calpain-1, a calcium-activated cysteine protease, which has been implicated in neuronal degeneration and death, was monitored by the proteolysis of spectrin in rat organotypic hippocampal slice cultures. Protease specific spectrin breakdown products revealed that LLP-1 generated~150/145-kDa fragments characteristic of calpain-1 activation in hippocampus undergoing cell death as evidenced by LDH release. This spectrin cleavage pattern was further confirmed by in vitro calpain-1 proteolysis. Futhermore, calpectin and MDL28170, inhibitors of calpain activity, blocked calpain-1-mediated spectrin cleavage. Spectrin cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin. Among pharmacological agents tested, apocynin, NADPH oxidase inhibitor, ameliorated the LLP-1-induced spectrin. Given the role of spectrin essential for synapse stabilization, LLP-1-induced spectrin cleavage as occurs with the activation of calpain-1 may be an important effector in LLP-1mediated cell injury in hippocampus, which is primarily linked to cognitive dysfunction.
Animals ; Calpain ; Cell Death ; Cysteine Proteases ; Hippocampus ; HIV* ; HIV-1* ; Humans* ; Lentivirus* ; NADPH Oxidase ; Neurons ; Protein Structure, Tertiary* ; Proteolysis ; Rats* ; Spectrin* ; Synapses ; Synaptophysin

RNA interference (RNAi) has exhibited huge potentials on anti-HIV-1 therapy research. The obtainment of RNAi element targeting to HIV-1 highly effectively and specifically was crucial for relevant research. Recent reports had described that microRNAs (miRNAs) posses more characteristics of inhibition and expression mechanisms than small interfering RNAs (siRNAs). In this study we explored the construction of artificial miRNA targeting to HIV-1 effectively and specifically. Sixteen siRNAs sequences were selected based on the conserved regions in the HIV-1 pol gene. ShRNA expression vectors were co-transfected with HIV-1 clone pNL4-3 to evaluate the abilities of siRNAs to inhibit HIV-1 expression. The pol1026 sequence was selected from candidates. The target sequence in the stem-loop structure of the well-characterized native miR-30a was replaced with pol1026 sequences, and the artificial miRNA expression vectors were co-transfected with the HIV-1 clone pNL4-3, results showed that HIV-1 can be effectively inhibited by miR-1026E. Target specificity of miR-1026E was confirmed by co-transfection assay with reporter plasmids containing different target sequences. The miR-1026E expression element was then inserted into Lentivirus which was used as a vector to transduce the MT-4 cells, MT-4-miR1026E expressing miR-1026E stably was cloned from transduced cells. The MT-4-miR1026E cell effectively inhibited HIV-1 replication in vitro. And the intracellular miR-181 and miR-16 expression levels and statl mRNA levels were not affected by the expression of miR-1026E in MT-4-miR1026E cells. miR-1026E is a promising candidate for future research.
Base Sequence ; Cloning, Molecular ; Gene Targeting ; methods ; Genetic Engineering ; Genetic Therapy ; methods ; HIV Infections ; virology ; HIV Protease ; genetics ; HIV-1 ; genetics ; physiology ; Lentivirus ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Molecular Sequence Data ; RNA Interference ; Transfection ; Virus Replication ; genetics

Effective and specific RNA interference (RNAi) elements are essential for the RNAi-based anti-HIV-1 research which has achieved extensive application. vif37 targeted to HIV-1 vi f is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase II promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37 H. The replication of HIV-1 was inhibited in MT-4-miR37 H cells which could express miR-vif37 H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37 H expression element. Real-time RT-PCR revealed that miR-vif37 H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not effected by the expression of miR-vif37 H in MT-4-miR37 H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.
Cell Line ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; metabolism ; HIV Infections ; virology ; HIV-1 ; genetics ; physiology ; Humans ; Lentivirus ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Nucleic Acid Conformation ; RNA Interference ; vif Gene Products, Human Immunodeficiency Virus ; chemistry ; genetics ; metabolism

As lentiviral vector holds the characteristics of higher transfection to non-dividing cells, larger capacity of transfer gene fragments, long-term expression of therapeutic gene and lower rate of immunological response, therefore it becomes potential viral vector in gene therapy. Improvements of lentiviral vector, human immunodeficiency virus type-I as example, and its application in gene transfer for gene therapy of hematological diseases are emphasized in this review.
Fanconi Anemia ; therapy ; Genetic Therapy ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; Hematologic Diseases ; therapy ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; metabolism ; Hemophilia A ; therapy ; Humans ; Lentivirus ; genetics ; Leukemia ; therapy ; beta-Thalassemia ; therapy

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.
Animals ; Catalytic Domain ; genetics ; Cattle ; DNA ; genetics ; DNA, Viral ; HIV-1 ; genetics ; metabolism ; Humans ; Immunodeficiency Virus, Bovine ; enzymology ; genetics ; Integrases ; chemistry ; genetics ; metabolism

Animals ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Rats

Gene therapy has been considered as one of the optimal treatments. Although, at the beginning of this century, a series of unexpected side effects brought gene therapy into depression, the improved lentiviral vectors, which characterised by high efficiency transfection, stable expression in target cells and good biosafety, have been applied in clinical trials in recent years and acquired a certain clinical improvements. Nowadays gene therapy becomes an eye-catching field. This review discusses the gene therapy how blocked by lentiviral vectors, the high efficiency and biosafety of lentiviral vectors, the improvement of lentiviral vector preparation and so on.
Animals ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Transfection

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.
Genetic Vectors ; HL-60 Cells ; Humans ; Lentivirus ; genetics ; Transfection