RESUMO
The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Anticorpos Neutralizantes , Anticorpos Anti-HIV/metabolismo , Glicosilação , Infecções por HIV/tratamento farmacológico , Polissacarídeos/metabolismoRESUMO
Viral infection generally induces polyclonal neutralizing antibody responses. However, how many lineages of antibody responses can fully represent the neutralization activities in sera has not been well studied. Using the newly designed stable HIV-1 Env trimer as hook, we isolated two distinct broadly neutralizing antibodies (bnAbs) from Chinese rhesus macaques infected with SHIV1157ipd3N4 for 5 years. One lineage of neutralizing antibodies (JT15 and JT16) targeted the V2-apex in the Env trimers, similar to the J038 lineage bnAbs identified in our previous study. The other lineage neutralizing antibody (JT18) targeted the V3 crown region in the Env, which strongly competed with human 447-52D. Each lineage antibody neutralized a different set of viruses. Interestingly, when the two neutralizing antibodies from different lineages isolated from the same macaque were combined, the mixture had a neutralization breath very similar to that from the cognate sera. Our study demonstrated that a minimum of two different neutralizing antibodies can fully recapitulate the serum neutralization breadth. This observation can have important implications in AIDS vaccine design.
Assuntos
Anticorpos Neutralizantes , Anticorpos Anti-HIV , HIV-1 , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios , Macaca mulatta/imunologia , Animais , HIV-1/imunologia , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Humanos , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/sangue , Vírus da Imunodeficiência Símia/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Testes de NeutralizaçãoRESUMO
Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.
Assuntos
Subunidade beta de Fator de Ligação ao Core , Infecções por HIV , HIV-1 , Produtos do Gene vif do Vírus da Imunodeficiência Humana , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Subunidade beta de Fator de Ligação ao Core/genética , HIV-1/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Linfócitos T/virologia , Replicação Viral , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
HIV remains a health challenge worldwide, partly because of the continued development of resistance to drugs. Therefore, it is urgent to find new HIV inhibitors and targets. Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3) are important host restriction factors that inhibit HIV-1 replication by their cytidine deaminase activity. HIV-1 viral infectivity factor (Vif) promotes proteasomal degradation of APOBEC3 proteins by recruiting the E3 ubiquitin ligase complex, in which core-binding factor ß (CBFß) is a necessary molecular chaperone. Interrupting the interaction between Vif and CBFß can release APOBEC3 proteins to inhibit HIV-1 replication and may be useful for developing new drug targets for HIV-1. In this study, we identified a potent small molecule inhibitor CBFß/Vif-3 (CV-3) of HIV-1 replication by employing structure-based virtual screening using the crystal structure of Vif and CBFß (PDB: 4N9F) and validated CV-3's antiviral activity. We found that CV-3 specifically inhibited HIV-1 replication (IC50 = 8.16 µm; 50% cytotoxic concentration >100 µm) in nonpermissive lymphocytes. Furthermore, CV-3 treatment rescued APOBEC3 family members (human APOBEC3G (hA3G), hA3C, and hA3F) in the presence of Vif and enabled hA3G packaging into HIV-1 virions, which resulted in Gly-to-Ala hypermutations in viral genomes. Finally, we used FRET to demonstrate that CV-3 inhibited the interaction between Vif and CBFß by simultaneously forming hydrogen bonds with residues Gln-67, Ile-102, and Arg-131 of CBFß. These findings demonstrate that CV-3 can effectively inhibit HIV-1 by blocking the interaction between Vif and CBFß and that this interaction can serve as a new target for developing HIV-1 inhibitors.
Assuntos
Desaminases APOBEC/metabolismo , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Subunidade beta de Fator de Ligação ao Core/metabolismo , HIV-1/efeitos dos fármacos , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.
Assuntos
HIV/genética , Lentivirus/genética , Transcrição Reversa/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Animais , Gatos , Bovinos , Células HEK293 , HIV/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Lentivirus/patogenicidade , Células Mieloides/virologia , Domínios Proteicos/genética , Proteína 1 com Domínio SAM e Domínio HD/química , Linfócitos T/virologia , Replicação Viral/genéticaRESUMO
One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFß E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFß interaction based on the sequences of Vif mutants with higher affinity for CBFß screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 µM and 55.1 µM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFß. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFß over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFß interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.
Assuntos
Fármacos Anti-HIV , Subunidade beta de Fator de Ligação ao Core , HIV-1 , Peptídeos , Ligação Proteica , Produtos do Gene vif do Vírus da Imunodeficiência Humana , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Humanos , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Células HEK293 , Subunidade beta de Fator de Ligação ao Core/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos/química , Fármacos Anti-HIV/farmacologia , Replicação Viral/efeitos dos fármacos , Desenho de Fármacos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismoRESUMO
Understanding maturation pathways of broadly neutralizing antibodies (bnAbs) against HIV-1 can be highly informative for HIV-1 vaccine development. A lineage of J038 bnAbs is now obtained from a long-term SHIV-infected macaque. J038 neutralizes 54% of global circulating HIV-1 strains. Its binding induces a unique "up" conformation for one of the V2 loops in the trimeric envelope glycoprotein and is heavily dependent on glycan, which provides nearly half of the binding surface. Their unmutated common ancestor neutralizes the autologous virus. Continuous maturation enhances neutralization potency and breadth of J038 lineage antibodies via expanding antibody-Env contact areas surrounding the core region contacted by germline-encoded residues. Developmental details and recognition features of J038 lineage antibodies revealed here provide a new pathway for elicitation and maturation of V2-targeting bnAbs.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Anticorpos Neutralizantes , Anticorpos Amplamente Neutralizantes , Epitopos , Anticorpos Anti-HIV , Humanos , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Non-human primates (NHP) are the only animal model suitable to evaluate the protection efficacy of HIV-1 vaccines. It is important to understand how and when neutralizing antibodies (nAbs) with specificities similar to those of human broadly neutralizing antibodies (bnAbs) develop in NHPs. To address these questions, we determined plasma neutralization specificities in two macaques which developed neutralization breadth after long-term simian/human immunodeficiency virus (SHIV) infection and identified neutralization escape mutations by analyzing the env sequences from longitudinal plasma samples. Neutralization activities targeting V2, CD4bs, V3 and gp120-gp41 interface only became detectable in week 350 plasma from macaques G1015R and G1020R using 25710 env mutants. When mapped with CAP45 env mutants, only V2 specificity was detected at week 217 and persisted until week 350 in G1015R. Neutralization escape mutations were found in CD4bs and V2 regions. However, all of them were different from those resistant mutations identified for human bnAbs. These results show that nAbs with specificities similar to human bnAbs are only detectable after long-term SHIV infection and that neutralization escape mutations in macaques are different from those found in HIV-1-infected individuals. These findings can have important implications in the best utilization of the NHP model to evaluate HIV-1 vaccines.