To analyze the variability and phenotype of envelope glycoprotein （Env） of human immunodeficiency virus type 1 （HIV-1） prevalent in Heilongjiang province, cloning of the full-length env gene from the peripheral bl...To analyze the variability and phenotype of envelope glycoprotein （Env） of human immunodeficiency virus type 1 （HIV-1） prevalent in Heilongjiang province, cloning of the full-length env gene from the peripheral blood mononuclear cells （PBMCs） of an HIV-1 positive individual in Heilongjiang province in China was performed by using conserved region primers. The amplified PCR products were cloned into a plasmid vector and sequenced. Phylogenetic analysis was done upon the full-length Env amino acid sequence. Subsequently, an HIV-1 pseudotyped virus bearing the envelope protein was constructed and the infectivity was examined using U87 cell lines expressing CD4 with either CCR5 or CXCR4. As the result, two functional env clones named as CHNHLJ03009c34 （GenBank Accession No： AY905493 ） and CHNHLJ03009c33 were obtained. It was found that the homology between CHNHLJ03009c34 and an HIV-1 subtype B＇ strain, RIA-2, isolated from Yunnan province, was 91.52% through comparing and analyzing full-length Env amino acid sequence of HIV-1 isolated from either China or abroad. Phylogenetic analysis indicated that CHNHLJ03009c34 has the closest molecular relation with strain RIA2 based on analyzing the full-length of the Env, while it became an independent branch upon analyzing the sequences of C2-V3 region of the Env. The secondary structure analysis of the envelope protein showed that the antigenicity and hydrophobicity of the strain demonstrated have no definite difference from that of RL42. Examination of infectivity showed that pseudovirus CHNHLI03009c34 could only infect U87. CD4. CCR5 cells, indicating that it was a RS-tropic HIV-1. In the conclusion, two HIV-1 env clones from an infected individual in Heilongjiang province have been identified as subtype B＇ and RS-tropic HIV-1. This is the first report on the analysis of primary isolates in Heilongjiang province.展开更多
The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) ...The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.展开更多
Human immunodeficiency virus type 1 （HIV-1）, a causative agent of AIDS, is affecting today more than 35 millions of people worldwide. The advance of anti-HIV chemotherapy has made AIDS a chronic non-fatal disease in...Human immunodeficiency virus type 1 （HIV-1）, a causative agent of AIDS, is affecting today more than 35 millions of people worldwide. The advance of anti-HIV chemotherapy has made AIDS a chronic non-fatal disease in resourceful countries. Longawaited anti-HIV-1 vaccine is still not with us yet; however, great progress in structural analyses of the envelope protein of HIV-1 in recent years starts to shed light on rational intervention targeted at the envelope protein, as will be reviewed in this article.展开更多
Even the potential of T cell-mimicking nanotrap for long term viral control due to its overcoming of human immunodeficiency virus(HIV)genetic diversity and viral resistance,the robust HIV inhibition was not expected b...Even the potential of T cell-mimicking nanotrap for long term viral control due to its overcoming of human immunodeficiency virus(HIV)genetic diversity and viral resistance,the robust HIV inhibition was not expected because these nanotraps displayed no obvious advantages compared with the infinite host cells.Herein,a glycoprotein 120(gp120)-targeting polypeptide UM15 reinforced lymphocyte-mimicking nanotrap was constructed,and its improved HIV-1 inhibiting efficacy was validated.According to the results,the constructed nanotraps exhibited evident escaping ability from uptake of the mononuclear phagocyte system and highly improved binding ability with gp120 proteins.The constructed nanotraps neutralized all tested HIV-1 pseudo typed viruses with IC80 of 21.0μg/mL,and inhibited both X4-tropic and R5-tropic HIV-1 with IC80 of 34.4 and 20.6μg/mL,respectively.Approximately 40%of gp120 was observed to be shed from pseudo virus,and above 40%bystander T cells were prevented from gp120-induced death by the constructed nanotraps.The safety of the constructed nanotraps was confirmed both in vitro and in mice.Therefore,the constructed nanotraps could specifically neutralize free HIV-1,selectively bind with gp120 expressing HIV-1 infected cells,cause gp120 shedding,inhibit gp120-induced bystander T cell killing on the premise of safety,and were considered as promising therapeutic agents for precise inhibition of HIV.展开更多
Human immunodeficiency virus-1 （HIV-1）encodes 15 viral proteins. Protein-protein interactions play a large role in the function of these proteins. In this study, we attempted to identify novel interactions between t...Human immunodeficiency virus-1 （HIV-1）encodes 15 viral proteins. Protein-protein interactions play a large role in the function of these proteins. In this study, we attempted to identify novel interactions between the HIV-1 proteins to better understand the role played by viral protein-protein interactions in the life cycle of HIV-I. Genes encoding the 15 viral proteins from the HIV-1 strain AD8 were inserted into the plasmids of a yeast two-hybrid system. By screening 120 pairs of proteins, interactions between seven pairs were found. This led to the discovery of an interaction between the HIV-1 proteins integrase （IN） and glycoprotein 41 （gp41）, which was confirmed by both co-immunoprecipitation （Co-IP） assays and fluorescence resonance energy transfer （FRET） imaging in live cells. In addition, it was found that the amino acids at positions 76-100 of gp41 are required for it to bind to IN. Deletion of this region from gp41 prevented its interaction with IN and reduced the production of HIV-1 in 293T cells. This study provides new information on HIV-1 protein-protein interactions which improves the understanding of the biological functions of gp41 and IN during the virus life cycle.展开更多
文摘To analyze the variability and phenotype of envelope glycoprotein （Env） of human immunodeficiency virus type 1 （HIV-1） prevalent in Heilongjiang province, cloning of the full-length env gene from the peripheral blood mononuclear cells （PBMCs） of an HIV-1 positive individual in Heilongjiang province in China was performed by using conserved region primers. The amplified PCR products were cloned into a plasmid vector and sequenced. Phylogenetic analysis was done upon the full-length Env amino acid sequence. Subsequently, an HIV-1 pseudotyped virus bearing the envelope protein was constructed and the infectivity was examined using U87 cell lines expressing CD4 with either CCR5 or CXCR4. As the result, two functional env clones named as CHNHLJ03009c34 （GenBank Accession No： AY905493 ） and CHNHLJ03009c33 were obtained. It was found that the homology between CHNHLJ03009c34 and an HIV-1 subtype B＇ strain, RIA-2, isolated from Yunnan province, was 91.52% through comparing and analyzing full-length Env amino acid sequence of HIV-1 isolated from either China or abroad. Phylogenetic analysis indicated that CHNHLJ03009c34 has the closest molecular relation with strain RIA2 based on analyzing the full-length of the Env, while it became an independent branch upon analyzing the sequences of C2-V3 region of the Env. The secondary structure analysis of the envelope protein showed that the antigenicity and hydrophobicity of the strain demonstrated have no definite difference from that of RL42. Examination of infectivity showed that pseudovirus CHNHLI03009c34 could only infect U87. CD4. CCR5 cells, indicating that it was a RS-tropic HIV-1. In the conclusion, two HIV-1 env clones from an infected individual in Heilongjiang province have been identified as subtype B＇ and RS-tropic HIV-1. This is the first report on the analysis of primary isolates in Heilongjiang province.
基金supported by grants from National Science and Technology Major Project(2012ZX10004701)
文摘The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.
文摘Human immunodeficiency virus type 1 （HIV-1）, a causative agent of AIDS, is affecting today more than 35 millions of people worldwide. The advance of anti-HIV chemotherapy has made AIDS a chronic non-fatal disease in resourceful countries. Longawaited anti-HIV-1 vaccine is still not with us yet; however, great progress in structural analyses of the envelope protein of HIV-1 in recent years starts to shed light on rational intervention targeted at the envelope protein, as will be reviewed in this article.
基金The current work was supported by the National Natural Science Foundation of China(No.81502675).
文摘Even the potential of T cell-mimicking nanotrap for long term viral control due to its overcoming of human immunodeficiency virus(HIV)genetic diversity and viral resistance,the robust HIV inhibition was not expected because these nanotraps displayed no obvious advantages compared with the infinite host cells.Herein,a glycoprotein 120(gp120)-targeting polypeptide UM15 reinforced lymphocyte-mimicking nanotrap was constructed,and its improved HIV-1 inhibiting efficacy was validated.According to the results,the constructed nanotraps exhibited evident escaping ability from uptake of the mononuclear phagocyte system and highly improved binding ability with gp120 proteins.The constructed nanotraps neutralized all tested HIV-1 pseudo typed viruses with IC80 of 21.0μg/mL,and inhibited both X4-tropic and R5-tropic HIV-1 with IC80 of 34.4 and 20.6μg/mL,respectively.Approximately 40%of gp120 was observed to be shed from pseudo virus,and above 40%bystander T cells were prevented from gp120-induced death by the constructed nanotraps.The safety of the constructed nanotraps was confirmed both in vitro and in mice.Therefore,the constructed nanotraps could specifically neutralize free HIV-1,selectively bind with gp120 expressing HIV-1 infected cells,cause gp120 shedding,inhibit gp120-induced bystander T cell killing on the premise of safety,and were considered as promising therapeutic agents for precise inhibition of HIV.
基金supported by the National Nano Project(grant no.2011CB933600)the National Natural Science Foundation of China(grant no.31470269)supported by the Institute of Biophysics and the National Laboratory of Biomacromolecules at the Chinese Academy of Sciences(CAS)
文摘Human immunodeficiency virus-1 （HIV-1）encodes 15 viral proteins. Protein-protein interactions play a large role in the function of these proteins. In this study, we attempted to identify novel interactions between the HIV-1 proteins to better understand the role played by viral protein-protein interactions in the life cycle of HIV-I. Genes encoding the 15 viral proteins from the HIV-1 strain AD8 were inserted into the plasmids of a yeast two-hybrid system. By screening 120 pairs of proteins, interactions between seven pairs were found. This led to the discovery of an interaction between the HIV-1 proteins integrase （IN） and glycoprotein 41 （gp41）, which was confirmed by both co-immunoprecipitation （Co-IP） assays and fluorescence resonance energy transfer （FRET） imaging in live cells. In addition, it was found that the amino acids at positions 76-100 of gp41 are required for it to bind to IN. Deletion of this region from gp41 prevented its interaction with IN and reduced the production of HIV-1 in 293T cells. This study provides new information on HIV-1 protein-protein interactions which improves the understanding of the biological functions of gp41 and IN during the virus life cycle.
