OBJECTIVE: To construct a HIV-1 gp120 transgenic mice (gp120 Tg) with vimentin (VIM) gene knockout. METHODS: Female HIV-1 gp120 Tg mice were mated to VIM heterozygote mice (F0). All the offspring mice were derived from these original founders so that both genotypes had the same mixed genetic background. The F1 mice were bred to generate of VIM, VIM, VIM/gp120 Tg and VIM/gp120 Tg mice. PCR was performed for genotyping of the mice, and the expressions of VIM and gp120 in the brain tissues were examined using immunoblotting. RESULTS: The results of PCR showed the presence of the target bands in VIM, VIM, VIM/gp120 Tg and VIM/gp120 Tg mice. In VIM/gp120 Tg mice, gp120 expression was detected throughout the brain regions while no VIM expression was detected. CONCLUSIONS We generated gp120 transgenic mouse models with VIM gene knockout, which facilitate the exploration of the role of VIM in gp120-induced neurotoxicity.
Animals ; Brain ; Disease Models, Animal ; Female ; HIV Envelope Protein gp120 ; HIV-1 ; Mice ; Mice, Knockout ; Mice, Transgenic ; Vimentin

OBJECTIVETo study the pathological changes of the liver tissues of patients with HIV infection.

METHODS14 biopsy and 12 autopsy liver tissues were examined histologically. HIV-1 related antigen of outer membrane protein gp120 and capsid protein p24 were examined with their corresponding monoclonal antibodies by immunohistochemistry.

RESULTSIn the biopsy group, cytomegalic virus (CMV) infection was found in one (1/14) case, outer membrane protein gp120 and/or capsid protein p24 antigen were detected in Kupffer cells and in some of the lymphocytes in 11 cases. All the hepatocytes were negative for outer membrane protein gp120 and capsid protein p24 antigens. In the autopsy group, there were 5 (5/12) cases of liver tissues with CMV infection and 5 cases each with mycobacterium and Toxoplasma gondii infection. Capsid protein p24 was detected in liver tissues in 3 cases.

CONCLUSIONThere is HIV infection in liver tissue of patients with HIV. The rate of opportunistic infections in liver biopsy samples was lower than that in the autopsy liver tissues of patients with HIV.

Adult ; Female ; HIV Core Protein p24 ; biosynthesis ; genetics ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; HIV Infections ; pathology ; Humans ; Liver ; pathology ; Male ; Middle Aged

OBJECTIVETo clone and express the HIV-1B gp120 genes isolated at different organizations from a patient died of AIDS dementia complex (ADC) in eukaryotic cells.

METHODSUsing the genomic DNA isolated from peripheral lymphnodes, choroid plexus and occipital white matter from a patient died of ADC as the template, HIV-1B gp120 gene was amplified with PCR. After sequenced, HIV-1B gp120 was inserted into pcDNA3.1 (+) and recombinant expressing vector gp120/pcDNA3.1 (+) was constructed succeffuly confirming with sequencing. Then expressing vector was transfected into eukaryotic cells U87 using liposome transfection and expression of HIV-1B gp120 gene was assayed with indirect immunofluorescence.

RESULTSHIV-1B gp120 genes isolated from peripheral lymphnodes, choroid plexus and occipital white matter of the ADC patient were successfully cloned and recombinant expressing vector gp120/pcDNA3; 1 (+) could express envelope glycoprotein HIV-1B gp120 in U87 cells.

CONCLUSIONAll the HIV-1B gp120 gene isolated at the different organizations of the same ADC patient could express in U87 cells, which may supply a valuable basis for studying the neurotoxicity and neurotoxic mechanism of HIV-1 gp120 protein.

AIDS Dementia Complex ; virology ; Cloning, Molecular ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; toxicity ; Humans ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

BACKGROUNDHuman immunodeficiency virus (HIV-1) is divided into two types, HIV-1 (groups M, N and O) and HIV-2. Heilongjiang Province located in the northeast of China, and the feature of the subtype distribution and sequence characteristics of HIV-1 strains prevalent in Heilongjiang Province is still uncertain. The aim of this study was to investigate the subtype distribution and genetic characteristics of HIV-1 strains in one hospital in Heilongjiang Province.

METHODSHIV-1 env gene was amplified by nested-PCR from peripheral blood mononuclear cells (PBMCs) obtained from 19 HIV-1 seropositive individuals in Heilongjiang Province. The C2-V3 region was sequenced. Aligned the nucleotide sequence of 19 samples with CLUSTAL W (BioEdit) software, results were acquired and used for phylogenetic tree analysis after artificial adjustment. Reference sequence, downloaded from Los Alamos HIV Sequence Database, was used to identify the subtype of obtained sequence. Genetic distance between sequences was assessed using the software MEGA 3.1 Kimura 2-parameter, and the Phylogenetic tree was reestablished with Neighbor-Joining method.

RESULTSPhylogenetic tree analysis showed that 19 Heilongjiang strains clustered closely to subtype B strain from Thailand and were far from other international subtype reference strains. Statistical test showed no significant discrepancy between the genetic distance of interclass and intra-class (P > 0.05). The analysis of V3 loop amino sequence of 19 Heilongjiang B strains revealed that V3 tip motif of 10 samples (52.63%) was GPGQ, and of 4 samples (21.53%) was GPGR.

CONCLUSIONSThe subtype of 19 HIV-1 seropositive individuals in Heilongjiang Province is B', and it is introduced from He'nan Province. V3 tip motifs of the HIV-1 isolates are mainly GPGQ and GPGR.

Adolescent ; Adult ; Amino Acid Sequence ; Female ; HIV Envelope Protein gp120 ; chemistry ; HIV-1 ; classification ; genetics ; Humans ; Male ; Molecular Sequence Data ; Peptide Fragments ; chemistry ; Phylogeny

OBJECTIVETo develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.

METHODSHL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.

RESULTSNo syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.

CONCLUSIONWe have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.

Biological Assay ; Cell Fusion ; Cell Line ; Coculture Techniques ; Drug Evaluation, Preclinical ; methods ; HIV Envelope Protein gp120 ; metabolism ; HIV Envelope Protein gp41 ; metabolism ; HIV Fusion Inhibitors ; chemistry ; pharmacology ; Humans ; beta-Galactosidase ; metabolism

OBJECTIVE: To explore the protective effect of SBi4211 (heptamidine), an inhibitor of S100B, against central nervous system injury induced by HIV-1 envelope protein gp120. METHODS: In an RESULTS: In the cell co-culture system, SBi4211 treatment significantly inhibited gp120-induced expression of S100B, RAGE and GFAP in U251 cells ( CONCLUSIONS SBi4211 can protect neurons from gp120-induced neurotoxicity possibly by inhibiting the S100B/ RAGE-mediated signaling pathway.
Animals ; Astrocytes ; Blotting, Western ; Central Nervous System ; HIV Envelope Protein gp120 ; Mice ; Neurons ; S100 Calcium Binding Protein beta Subunit ; Signal Transduction

OBJECTIVETo study the characteristics of human immunodeficiency virus (HIV-1) V3 loop amino acid mutations among HIV-1 infected people in Liaoning province.

METHODSThe whole blood samples of the HIV carriers and AIDS patients were collected in Liaoning province, China and were extracted PBMC genome DNA. HIV-1 V3 and flanking region sequences were amplified by nest-polymerase chain reaction (nest-PCR) with env specific primers: ED5/ED12 and ED31/ED33. Products were sequenced directly and sequences were aligned, translated and analyzed.

RESULTSIn AIDS group, some amino acid mutations at specific position of V3 loop: S to R at position 11, H to S, T and N at position 13, A to V at position 19, F to Y at position 20, Q or D to N at position 25 and 29, were found and all common mutations were associated with T tropic/SI phenotype. The frequency of such amino acid mutations in specific positions was higher in AIDS group than that of the asymptomatic infection group (P < 0.05). In addition, we found some unusual tetramer compositions on the tip of V3 loop: GQGR, APGR and RPGA, GLGR, RPGA in addition to some rare mutations, such as: N to H at position 5 and H to S, F at position 34.

CONCLUSIONThe amino acid mutations on the V3 loop of HIV-1 epidemic in Liaoning province were in agreement with the results of subtype B, but we observed some rare mutations and unusual tetramer compositions on the tip of V3 loop.

Acquired Immunodeficiency Syndrome ; epidemiology ; virology ; Adult ; Amino Acid Sequence ; Child ; China ; epidemiology ; Female ; Genes, env ; genetics ; HIV Envelope Protein gp120 ; genetics ; HIV Infections ; epidemiology ; virology ; HIV-1 ; classification ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Viral Envelope Proteins ; genetics

BACKGROUNDThe adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.

METHODSMost circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.

RESULTSResponse of gp120-specific antibody was relatively low after DNA primes (mean titer = 10(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer = 10(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P < 0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.

CONCLUSIONPolyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.

AIDS Vaccines ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Female ; HIV Envelope Protein gp120 ; genetics ; immunology ; Humans ; Immunization ; Immunoglobulin G ; blood ; Phylogeny ; Rabbits ; Vaccines, DNA ; immunology

OBJECTIVENew rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China.

METHODSThe antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells.

RESULTSWe found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67.

CONCLUSIONHydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.

Amino Acid Sequence ; Anti-HIV Agents ; chemistry ; pharmacology ; China ; epidemiology ; HIV Envelope Protein gp120 ; genetics ; metabolism ; HIV Infections ; epidemiology ; virology ; HIV-1 ; drug effects ; genetics ; Humans ; Peptides, Cyclic ; administration & dosage ; pharmacology ; Phylogeny

OBJECTIVETo screen the HIV-1 entry inhibitors targeting HIV-1 gp120 from the IBS natural product database by virtual screening based on the binding mode of the neutralizing antibody VRC01 with HIV-1 gp120 and investigate the anti-viral activities of the inhibitors and their action mechanisms.

METHODSThe binding interaction of the candidate molecules binding gp120 and changes of the binding free energy were analyzed by MM-PBSA calculation. The anti-HIV-1 activities of the tested compounds were detected by HIV-1 pseudotyped virus, laboratory-adapted HIV-1 and a cell-cell fusion assay. The cytotoxicity of the studied molecules was examined by XTT colorimetric assay. The mechanisms of the anti-viral activities of the candidate molecules were analyzed using enzyme-linked immunosorbent assay.

RESULTSA total of 19 molecules with distinct reduction of the binding free energy after binding with gp120 were screened from 40000 molecules. Among them, NC-2 showed anti-HIV-1 activities against HIV-1 pseudotyped virus and laboratory-adapted HIV-1, and was capable of blocking HIV-1 envelope-mediated cell-cell fusion. The IC50 of NC-2 for inhibiting HIV-1IIIB and pseudotyped HIV-1JRFL infection were 1.95∓0.44 µmol/L and 10.58∓0.13 µmol/L, respectively. The results of ELISA suggested that NC-2 could inhibit the binding of HIV-1 gp120 to CD4 without blocking the formation of gp41 six-helix bundle in vitro.

CONCLUSIONThis computer-based virtual screening method can be used to screen HIV-1 entry inhibitors targeting gp120. Using this virtual screening approach combined with anti-viral activity screening, we obtained a potent HIV-1 entry inhibitor NC-2 with novel structure.

Anti-HIV Agents ; pharmacology ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Neutralizing ; pharmacology ; Binding Sites ; Cell Fusion ; Cell Line ; Drug Discovery ; Drug Evaluation, Preclinical ; HIV Antibodies ; pharmacology ; HIV Envelope Protein gp120 ; antagonists & inhibitors ; HIV-1 ; drug effects ; Humans ; Microbial Sensitivity Tests