BACKGROUNDConstruction of replication-deficient recombinant adenovirus expressing gag-pol and env genes of human immunodeficiency virus (HIV) in mice.

METHODSgag-polDelta and gp140TM genes were cloned into shuttle vector pAdTrack-CMV respectively, and then the plasmids containing gag-polDelta or gp140TM gene were cotransformed with the backbone of adenovirus into E.coli BJ5183. Transfections of the recombinants were performed to obtain recombinant adenoviruses. Its immunogenicity was evaluated by testing antibody levels of mice primed with DNA vaccines and boosted with recombinant adenoviruses.

RESULTSThe replication-deficient recombinant adenovirus could express Gp140TM, Gag P55 and P24 proteins correctly. The mice primed with DNA vaccines and boosted with recombinant adenoviruses elicited high titer of HIV-1-specific antibody compared with that inoculated with DNA vaccines only.

CONCLUSIONReplication-deficient recombinant adenovirus expressing gag-polDelta and gp140TM can elicit high titer HIV-1-specific antibodies.

AIDS Vaccines ; immunology ; Adenoviridae ; genetics ; Animals ; Female ; Fusion Proteins, gag-pol ; biosynthesis ; genetics ; Gene Products, env ; biosynthesis ; genetics ; HIV-1 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; Transfection ; Vaccines, DNA ; immunology ; env Gene Products, Human Immunodeficiency Virus

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
Anti-HIV Agents ; pharmacology ; Benzoxazines ; pharmacology ; Bioluminescence Resonance Energy Transfer Techniques ; methods ; Fusion Proteins, gag-pol ; genetics ; metabolism ; HEK293 Cells ; HIV Protease ; metabolism ; physiology ; HIV-1 ; enzymology ; High-Throughput Screening Assays ; methods ; Humans ; Plasmids ; genetics ; Protein Precursors ; metabolism ; physiology ; Pyridazines ; pharmacology ; Transfection ; Virion ; growth & development ; Virus Assembly ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

OBJECTIVETo investigate the subtype distribution and the prevalence of sequence characteristics of HIV-1 strains in Beijing residents during 2006 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routines.

METHODSBlood samples from 32 new confirmed HIV-1 infected individuals from Beijing residents in 2006 and separated plasma specimens were collected. RNAs were extracted and the gag and env gene were amplified by RT-PCR and nest-PCR. PCR products were sequenced directly and phylogenetic analyses of gag and env gene were performed using the MEGA2 software.

RESULTSAmong 32 HIV-1 plasma samples, 22 gag and 4 env gene fragments were amplified and analyzed. Five HIV-1 subtypes or circulating recombinant forms(CRFs) of HIV-1 including Thai B (2 strains), B (9 strains), C (2 strains), CRF07_BC (5 strains), CRF01 AE (4 strains) were identified being circulated in Beijing. The gene divergences of gag gene inside the subtypes were 6.6%, 4.3%, 6.8%, 4.9% and 3.0% in subtype B, Thai B, C, CRF01_AE and CRF07_BC respectively. Subtypes B were predominant in Beijing, accounted for 40.9% among 22 samples.

CONCLUSIONFive HIV-1 subtypes were identified in Beijing and the surveillance of HIV-1 gene variation should be paid more attention to.

China ; HIV-1 ; classification ; genetics ; Humans ; Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; env Gene Products, Human Immunodeficiency Virus ; genetics ; gag Gene Products, Human Immunodeficiency Virus ; genetics

The 109 whole blood samples were collected from HIV-1 infected former blood donors in Henan and Shanxi. The RNA templates were extracted from plasma and used for the full gag gene amplification and sequencing. The sequences were divided into 3 groups according to sampling year. The Entropy software was used to identify the amino acids with composition difference among different groups of amino acid sequences. The results showed that there existed 8 and 13 amino acid sites with the statistical significance difference, respectively, in sequences in year 2004 and 2005, compared to those in 2002. Among them, there existed 5 amino acid sites in two groups. Of 16 amino acid sites, the increasing polymorphism and the decreasing polymorphism along the sampling year were observed in 10 and 6 amino acid sites respectively. Of 10 sites with increased polymorphism, 8 sites were located in the CTL epitopes recognized and presented by the main HLA alleles existed in Chinese population. The 6 sites with decreasing polymorphism all existed in main domains of Gag proteins.
Blood Donors ; China ; epidemiology ; Genetic Variation ; HIV-1 ; genetics ; Humans ; Polymorphism, Genetic ; gag Gene Products, Human Immunodeficiency Virus ; genetics

To investigate the subtype distribution of human immunodeficiency virus-1(HIV-1) infection among men who have sex with men (MSM) in Zhengzhou, Henan Province, forty blood samples were collected from HIV-1 carriers, who acknowledged to have sex with men. The complete gag gene was amplified by RT-PCR and nested-PCR and sequenced. All sequences were edited by BioEdit and subtyped by genotyping software. Phylogenetic analysis of gag gene were then performed using the MEGA 3.1 software, the gene distances were calculated by Distance program. There were three different HIV-1 subtypes including B, CRF01-AE and CRF07-BC present among twenty four MSMs in Zhengzhou. Genotyping results showed that 33.33% (8/24) were B, 41.67% (10/24) were CRF01-AE and 25% (6/24) were CRF07-BC, and subtype CRF01-AE had become the most prevalent HIV-1 subtype in Zhengzhou, Henan province. In conclusion, recombinant HIV-1 strains are circulating in Henan province and the epidemiology is complicated.
Adult ; China ; HIV-1 ; classification ; genetics ; isolation & purification ; Homosexuality, Male ; Humans ; Male ; Middle Aged ; Phylogeny ; Sequence Analysis, DNA ; Young Adult ; gag Gene Products, Human Immunodeficiency Virus ; genetics

OBJECTIVETo estimate prevalence of HIV/AIDS among children and the transmission routes in a highly endemic villages of AIDS.

METHODSTotally 208 high-risk women of child bearing age and 159 of their children aged 0 - 14 years were investigated. Their medical histories of blood donation or transfusion were collected, blood samples were taken and sera were separated for HIV test. Enzyme-linked immunosorbent assay (ELISA) and Western blot assay were performed for HIV antibody. The Nested-polymerase chain reaction (PCR) assay amplifying gag gene p17 was performed on samples of children aged less than 18 months.

RESULTSThirty-seven HIV infected cases were found among 159 children aged 0 - 14 years of whom 33 were infected by mother-to-child transmission (89.2%, 33/37), 3 by blood transfusion (8.1%, 3/37) and one by iatrogenic route (2.7%, 1/37). Sixty seven mothers who were seropositive for HIV and their 86 children who were born after 1992 were investigated, 33 cases of them were infected with HIV. The rate of vertical transmission was 38.4% (33/86). The HIV vertical transmission rate among mothers with AIDS (68.8%, 22/32) was significantly greater than that among mothers with asymptomatic HIV infection (20.4%, 11/54, P < 0.05). The number of children infected with HIV through vertical transmission increased from 1993 to 2001. Among 37 children infected with HIV, 12 cases developed AIDS and 4 of them died, of whom 2 cases died from tuberculosis. The morbidity of AIDS was 27.3% (9/33). Ninety three point nine percent (31/33) of infected mothers didn't know their HIV seropositive status before pregnancy and delivery. Of 8 pregnant women infected with HIV, one had aggravation of AIDS, 2 miscarried, 2 terminated their pregnancy and 3 continued their pregnancy.

CONCLUSIONMother-to-child transmission of HIV was the major route of HIV/AIDS transmission to the children. The main reason leading to HIV infection in children was the lack of prenatal HIV counseling and testing for the high-risk women of childbearing age and lake of interventions. The countermeasures must be taken to control the further transmission of AIDS in order to protect the health of women and children in the highly endemic areas of AIDS.

Acquired Immunodeficiency Syndrome ; diagnosis ; epidemiology ; transmission ; Adolescent ; Adult ; Antibodies, Viral ; blood ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Gene Products, gag ; genetics ; HIV Antigens ; genetics ; HIV Infections ; diagnosis ; epidemiology ; transmission ; HIV-1 ; genetics ; immunology ; Humans ; Infant ; Infectious Disease Transmission, Vertical ; Male ; Polymerase Chain Reaction ; Prevalence ; Viral Proteins ; gag Gene Products, Human Immunodeficiency Virus

BACKGROUNDDeveloping an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy.

METHODSWe constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy.

RESULTSOur data demonstrated that the rTTV-lucgag was able to express high level of luciferase (< or = 10(6) relative luciferase units (RLU)/mg protein) and HIV-1 Gag (> 3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r(2) = 0.71, P < 0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006 +/- 3141) RLU/mg protein in control group to (1538 +/- 463) RLU/mg protein in vaccine group (P = 0.1969).

CONCLUSIONSThe luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

AIDS Vaccines ; genetics ; Animals ; Female ; HIV Infections ; immunology ; prevention & control ; HIV-1 ; genetics ; Humans ; Kinetics ; Luciferases ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Poxviridae ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Virus Replication ; genetics ; gag Gene Products, Human Immunodeficiency Virus ; genetics

To investigate the effects of induction temperature on the expression product and the impact of urea concentration on the purification, HIV-1 Gag inclusion bodies from E. coli induced at 30 degrees C (IB30) and 37 degrees C (IB37) were dissolved with urea of different concentrations. The solubility and yield of refolding were compared. IB30 were dissolved with 2 mol/L and 8 mol/L urea, and then purified with chromatography. IB30 were found easier to be solubilized in low concentration of urea and easier to be refolded than IB37. Furthermore, compared to the IB30 dissolved in 8 mol/L urea, Gag protein solubilized in 2 mol/L urea was purified to higher purity with gel filtration (GF) and ion exchange (IEX) chromatography. Gag inclusion body induced at lower temperature may contain more protein with native-like or reversibly-denatured structures, and solubilization in the presence of low concentrations of urea can help to retain these structures. This study has provided new insights into the purification of proteins from inclusion bodies.
Escherichia coli ; genetics ; metabolism ; HIV-1 ; genetics ; Humans ; Inclusion Bodies ; metabolism ; Protein Denaturation ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Temperature ; Urea ; chemistry ; gag Gene Products, Human Immunodeficiency Virus ; biosynthesis ; genetics

To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent in Beijing during 2007 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routes, we collected the anti-conglutinated whole blood samples from HIV-1 newly infected individuals in Beijing during 2007 and separated plasma specimens from the aamples. RNAs were extracted and the gag genes from the various samples were amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag genes were performed using the MEGA2 software. Among 200 HIV-1 plasma samples,161 gag HIV-1 gene fragments were amplified and analyzed. Seven HIV-1 subtypes or circulating recombinant forms (CRFs) of HIV-1 including A1 (1 strains), B (35 strains), Thai B (19 strains), C (3 strains), CRF01_AE (49 strains), CRF07_BC (51 strains), CRF08_BC (3 strain) were identified circulating in Beijing. The gene divergences inside the subtypes were different, with 7.7%, 6.5%, 6.7%, 6.7%, 5.5%, 4.3%, 5.8%, in subtype A1, B, Thai B, C, CRF01_AE, CRF07_BC, CRF08_BC, respectively. Subtypes CRF07_BC and CRF01_AE were predominant in Beijing account for 31.7% and 30.4% among samples. Seven HIV-1 subtypes exist in Beijing and the surveillance of HIV-1 gene variation should be paid more attention.
Adolescent ; Adult ; China ; epidemiology ; Genotype ; HIV Infections ; epidemiology ; virology ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Molecular Epidemiology ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Young Adult ; gag Gene Products, Human Immunodeficiency Virus ; genetics

The late stages of the HIV-1 replication cycle are important to the overall replication cycle. During the late stages, HIV-1 replication undergoes the processes of assembly, release, and maturation, resulting in the production of a mature virus particle capable of infecting a new target cell. The structural protein Gag and its related gene (protein) play a central role in these pathways. The different regions of Gag worked in concert to drive production of a mature infectious particle through protein-protein, protein-RNA and protein-lipid interactions. The designed drug aimed directly at these stages can efficiently block the maturation and infectivity of HIV-1. In this article, the role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and related inhibitors is reviewed.
Amphotericin B ; analogs & derivatives ; chemistry ; pharmacology ; Anti-HIV Agents ; chemistry ; pharmacology ; Benzeneacetamides ; chemistry ; pharmacology ; Furans ; chemistry ; pharmacology ; Genes, gag ; HIV-1 ; drug effects ; physiology ; Humans ; Phenylurea Compounds ; chemistry ; pharmacology ; Piperidines ; chemistry ; pharmacology ; Succinates ; chemistry ; pharmacology ; Sulfur Compounds ; chemistry ; pharmacology ; Triterpenes ; chemistry ; pharmacology ; Virus Assembly ; drug effects ; Virus Release ; drug effects ; Virus Replication ; drug effects ; physiology ; gag Gene Products, Human Immunodeficiency Virus ; metabolism ; physiology