RESUMO
Two Gordonia bacteriophages, GrootJr and NovumRegina, were discovered, sequenced, and annotated. These phages were isolated from distinct soil and water samples, respectively, on Gordonia terrae 3612. These phages belong to the CR2 subcluster and are similar in genome size and gene content.
RESUMO
Two mycobacteriophage genomes were newly sequenced and annotated. Duggie and Hocus were discovered, enriched, and isolated from soil using Mycobacterium smegmatis mc2155. The bacteriophages are lytic Siphoviridae and belong to the B1 subcluster. The Hocus and Duggie genomes are highly similar to one another in both nucleotide sequence and gene content.
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Three mycobacteriophages, Bipolarisk, Bread, and FudgeTart, were isolated from enriched soil samples found in Crete, NE. All three phages are lytic, belong to subcluster C1, and infect Mycobacterium smegmatis mc2155. The structures of the three genomes are similar, with slight variations in gene number and content.
RESUMO
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
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.
Assuntos
Proteína gp120 do Envelope de HIV/química , HIV-1/fisiologia , HIV-2/fisiologia , Multimerização Proteica , Vírus da Imunodeficiência Símia/fisiologia , Internalização do Vírus , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-2/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/genética , Estabilidade Proteica , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genéticaRESUMO
The twin-cysteine motif (TCM) in the V2 loop region of gp120, identified in our previous report on the simian immunodeficiency virus mac239 (SIVmac239), is a conserved evolutionary element in all primate lentiviruses except for HIV-1 which has lost the TCM during cross-species transmission. In this study, we have further explored the TCM in other SIV and HIV-2 strains. Our data shows that strains from different evolutionary lineages have different phenotypes when the twin-cysteines are removed. In the SIVsm/HIV-2 lineage, removal of the twin-cysteines decreases envelope trimer stability, but in the SIVagm lineage, a blockage of gp160 processing is observed. Molecular modeling has confirmed that the twin-cysteines do form a disulfide bond in the gp120 subunit, which interacts with the V1 loop to stabilize the envelope trimer. Therefore, we hypothesize that if the TCM is added back to HIV-1, it will enhance envelope stability for vaccine immunogen design.
Assuntos
Motivos de Aminoácidos , Cisteína/química , Proteína gp120 do Envelope de HIV/química , HIV-1/química , HIV-2/química , Vírus da Imunodeficiência Símia/química , Proteínas do Envelope Viral/química , Vacinas contra a AIDS , Sequência de Aminoácidos , Animais , Linhagem Celular , Cisteína/genética , Desenho de Fármacos , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-2/genética , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genéticaRESUMO
The outer-domain core of gp120 may serve as a better HIV vaccine immunogen than the full-length gp120 because of its greater stability and immunogenicity. In our previous report, we introduced two disulfide bonds to the outer-domain core of gp120 to fix its conformation into a CD4-bound state, which resulted in a significant increase in its immunogenicity when compared to the wild-type outer-domain core. In this report, to further improve the immunogenicity of the outer-domain core based immunogen, we have introduced a Tryptophan residue at gp120 amino acid sequence position 375 (375S/W). Our data from immunized guinea pigs indeed shows a striking increase in the immune response due to this stabilized core outer-domain. Therefore, we conclude that the addition of 375W to the outer-domain core of gp120 further stabilizes the structure of immunogen and increases the immunogenicity.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Imunogenicidade da Vacina , Triptofano/química , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/química , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Antígenos CD4 , Desenho de Fármacos , Epitopos/química , Cobaias , Anticorpos Anti-HIV/sangue , HIV-1/imunologiaRESUMO
Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter ß-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture.
Assuntos
Anticorpos Monoclonais/metabolismo , Escherichia coli/metabolismo , Anticorpos Anti-HIV/metabolismo , HIV-1/fisiologia , Anticorpos de Cadeia Única/fisiologia , Anticorpos Neutralizantes/fisiologia , Sítios de Ligação , Anticorpos Amplamente Neutralizantes , Antígenos CD4 , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Modelos Moleculares , Neisseria gonorrhoeae , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 ("twin cysteines") is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV(cpz) lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV(cpz) as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.
Assuntos
Cisteína/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Multimerização Proteica , Vírus da Imunodeficiência Símia/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Fusão Celular , Linhagem Celular , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Filogenia , Estabilidade Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Receptores Virais/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Relação Estrutura-AtividadeRESUMO
Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses.
