RESUMO
Scytovirin (SVN) is a lectin from cyanobacteria which has a strong inhibitory activity against Ebola virus infection. We engineered scytovirin as the inhibitor for surface display of lactic acid bacteria to block Ebola virus infection. Two different bacterial strains (Lactobacillus casei and Lactococcus lactis) were successfully engineered for scytovirin expression on the bacterial surface. These bacteria were found to be effective at neutralizing pseudotyped Ebolavirus in a cell-based assay. This approach can be utilized for prophylactic prevention, as well as for treatment. Since lactic acid bacteria can colonize the human body, a long-term efficacy could be achieved. Furthermore, this approach is also simple and cost-effective and can be easily applied in the regions of Ebola outbreaks in the developing countries.
RESUMO
Membrane proteins expressed on the surface of enveloped viruses are conformational antigens readily recognized by B cells of the immune system. An effective vaccine would require the synthesis and delivery of these native conformational antigens in lipid membranes that preserve specific epitope structures. We have created an extracellular vesicle-based technology that allows viral membrane antigens to be selectively recruited onto the surface of WW domain-activated extracellular vesicles (WAEVs). Budding of WAEVs requires secretory carrier-associated membrane protein 3, which through its proline-proline-alanine-tyrosine motif interacts with WW domains to recruit fused viral membrane antigens onto WAEVs. Immunization with influenza and HIV viral membrane proteins displayed on WAEVs elicits production of virus-specific neutralizing antibodies and, in the case of influenza antigens, protects mice from the lethal viral infection. WAEVs thus represent a versatile platform for presenting and delivering membrane antigens as vaccines against influenza, HIV, and potentially many other viral pathogens.
Assuntos
Vesículas Extracelulares , Infecções por HIV , Vacinas contra Influenza , Influenza Humana , Animais , Camundongos , Humanos , Antígenos Virais , Domínios WW , Antígenos , ProlinaRESUMO
Filoviruses enter cells through macropinocytosis and trafficking into the endosomes in which they bind to the receptor Niemann-Pick C1 protein (NPC1) for membrane fusion and entry into the cytoplasm. The endosomal receptor-binding is critical step for filovirus entry. Designing inhibitors to block receptor binding will prevent viral entry. Using available binding structural information from the co-crystal structures of the viral GP with the receptor NPC1 or with monoclonal antibodies, we have conducted structure-based design of peptide inhibitors to target the receptor binding site (RBS). The designed peptides were tested for their inhibition activity against pseudo-typed or replication-competent viruses in a cell-based assay. The results indicate that these peptides exhibited strong activities against both Ebola and Marburg virus infection. It is expected that these peptides can be further developed for therapeutic use to treat filovirus infection and combat the outbreaks.
Assuntos
Filoviridae , Receptores Virais , Inibidores de Proteínas Virais de Fusão , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular , Ebolavirus/fisiologia , Endossomos/metabolismo , Filoviridae/química , Filoviridae/efeitos dos fármacos , Doença pelo Vírus Ebola , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Glicoproteínas de Membrana/metabolismo , Proteína C1 de Niemann-Pick/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Inibidores de Proteínas Virais de Fusão/química , Inibidores de Proteínas Virais de Fusão/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Lactobacillus bacteria are potential delivery vehicles for biopharmaceutical molecules because they are well-recognized as safe microorganisms that naturally inhabit the human body. The goal of this study was to employ these lactobacilli to combat human immunodeficiency virus type 1 (HIV-1) infection and transmission. By using a chromosomal integration method, we engineered Lactobacillus acidophilus ATCC 4356 to display human CD4, the HIV-1 receptor, on the cell surface. Since human CD4 can bind to any infectious HIV-1 particles, the engineered lactobacilli can potentially capture HIV-1 of different subtypes and prevent infection. Our data demonstrate that the CD4-carrying bacteria are able to adsorb HIV-1 particles and reduce infection significantly in vitro and also block intrarectal HIV-1 infection in a humanized mouse model in preliminary tests in vivo Our results support the potential of this approach to decrease the efficiency of HIV-1 sexual transmission.IMPORTANCE In the absence of an effective vaccine, alternative approaches to block HIV-1 infection and transmission with commensal bacteria expressing antiviral proteins are being considered. This report provides a proof-of-concept by using Lactobacillus bacteria stably expressing the HIV-1 receptor CD4 to capture and neutralize HIV-1 in vitro and in a humanized mouse model. The stable expression of antiviral proteins, such as CD4, following genomic integration of the corresponding genes into this Lactobacillus strain may contribute to the prevention of HIV-1 sexual transmission.
Assuntos
Antígenos CD4/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/metabolismo , Lactobacillus acidophilus/metabolismo , Animais , Antígenos CD4/genética , Linhagem Celular , Feminino , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , Humanos , Lactobacillus acidophilus/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , HIV-1/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Vírion/metabolismo , Vírion/fisiologia , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein (Env) becomes exposed after CD4 binding and contacts the coreceptor to mediate viral entry. Prior to CD4 engagement, a hydrophobic patch located at the tip of the V3 loop stabilizes the non-covalent association of gp120 with the Env trimer of HIV-1 subtype B strains. Here, we show that this conserved hydrophobic patch (amino acid residues 307, 309 and 317) contributes to gp120-trimer association in HIV-1 subtype C, HIV-2 and SIV. Changes that reduced the hydrophobicity of these V3 residues resulted in increased gp120 shedding and decreased Env-mediated cell-cell fusion and virus entry in the different primate immunodeficiency viruses tested. Thus, the hydrophobic patch is an evolutionarily conserved element in the tip of the gp120 V3 loop that plays an essential role in maintaining the stability of the pre-triggered Env trimer in diverse primate immunodeficiency viruses.