scholarly journals Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

2013 ◽  
Vol 57 (10) ◽  
pp. 4743-4750 ◽  
Author(s):  
Mark Contarino ◽  
Arangassery R. Bastian ◽  
Ramalingam Venkat Kalyana Sundaram ◽  
Karyn McFadden ◽  
Caitlin Duffy ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Gel Filtration ◽  
Etiologic Agent ◽  
Host Cells ◽  
Dependent Manner ◽  
Cell Infection ◽  
C Terminus ◽  
P24 Protein ◽  
Env Protein ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)xlinker (wherexis 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.

scholarly journals Effects of Gag Mutation and Processing on Retroviral Dimeric RNA Maturation

2006 ◽  
Vol 80 (3) ◽  
pp. 1242-1249 ◽  
Author(s):  
William Fu ◽  
Que Dang ◽  
Kunio Nagashima ◽  
Eric O. Freed ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Viral Protein ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Protein Cleavage ◽  
Rna Packaging ◽  
Virus Rna ◽  
Rna Dimer ◽  
Rna Maturation ◽  
Hiv 1

ABSTRACT After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.

scholarly journals Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles

2005 ◽  
Vol 79 (2) ◽  
pp. 834-840 ◽  
Author(s):  
Christoph A. Merten ◽  
Jörn Stitz ◽  
Gundula Braun ◽  
Eric M. Poeschla ◽  
Klaus Cichutek ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cleavage Site ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Hiv Protease ◽  
Evolution System ◽  
Peptide Cleavage ◽  
C Terminus ◽  
Hiv 1 ◽  
Vector Particles

ABSTRACT In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

scholarly journals SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System

2005 ◽  
Vol 73 (7) ◽  
pp. 4327-4337 ◽  
Author(s):  
Kristen J. Kanack ◽  
J. Adam Crawford ◽  
Ichiro Tatsuno ◽  
Mohamed A. Karmali ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Bacterial Attachment ◽  
Host Cells ◽  
Dependent Manner ◽  
Cell Infection ◽  
Type Iii

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

scholarly journals Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations

1998 ◽  
Vol 72 (12) ◽  
pp. 9621-9627 ◽  
Author(s):  
Rosemary E. Kiernan ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.

scholarly journals HIV-1 P24 EXPRESSION IN TRANSPLASTOMIC TOBACCO PLANTS

2020 ◽  
Vol 15 (2) ◽  
pp. 52-68
Author(s):  
Loc Tuong Phan ◽  
Ho Huu Nguyen ◽  
Thanh Thi Nguyen
Keyword(s):  
Western Blot ◽  
Southern Blot ◽  
Gel Filtration ◽  
Target Site ◽  
Gel Filtration Chromatography ◽  
Tobacco Plants ◽  
Transplastomic Tobacco ◽  
P24 Protein ◽  
High Concentrations ◽  
Hiv 1

Expression of HIV-1 p24 gene in chloroplasts was achieved in a tobacco variety V2 (Virginia TBE2). Through PCR and Southern blot analyses, it was demonstrated that the transgene integrated into the target site in the chloroplasts, between trnfM and trnG. Western blot results showed that HIV-1 p24 gene expressed in transplastomic tobacco plants. p24 protein accumulations were detected by ELISA in the range from 1.7% to 6.3% TSP and the high concentrations in the leaves near the top. p24 protein was purified by gel filtration chromatography demonstrated that the purification is 9.694 folds and the performance is 31.94%, however, protein p24 largely was inactive after purification.

scholarly journals Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter ofTrypanosoma cruzi

2019 ◽  
Vol 30 (14) ◽  
pp. 1676-1690 ◽  
Author(s):  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Mayara S. Bertolini ◽  
Anibal E. Vercesi ◽  
Roberto Docampo
Keyword(s):  
Citrate Synthase ◽  
Etiologic Agent ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Amino Acid Residues ◽  
Cell Infection ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Low Glucose ◽  
Membrane Spanning

We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc and TcMCUd. The predicted structure of the proteins indicates that, as predicted for the TcMCU and TcMCUb paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca2+uptake, while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca2+uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca2+uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared with those in the mammalian complex.

APOBEC3B potently restricts HIV-2 but not HIV-1 in a Vif-dependent manner

2021 ◽  
Author(s):  
Susana Bandarra ◽  
Eri Miyagi ◽  
Ana Clara Ribeiro ◽  
João Gonçalves ◽  
Klaus Strebel ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Functional Adaptation ◽  
Host Cells ◽  
Cancer Therapies ◽  
Dependent Manner ◽  
Induced Mutations ◽  
Cytidine Deaminases ◽  
Protein Functions ◽  
Human Viruses ◽  
Hiv 1

Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2 but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partner’s in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif -encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

scholarly journals Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

2020 ◽  
Vol 295 (37) ◽  
pp. 13023-13030
Author(s):  
Seiya Ozono ◽  
Yanzhao Zhang ◽  
Minoru Tobiume ◽  
Satoshi Kishigami ◽  
Kenzo Tokunaga
Keyword(s):  
Virus Production ◽  
Integrase Inhibitor ◽  
Luciferase Assay ◽  
Protein Levels ◽  
C Terminus ◽  
P24 Protein ◽  
Luminescent Signal ◽  
Routine Handling ◽  
Hiv 1 ◽  
Peptide Tag

In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.

scholarly journals New Connections: Cell-to-Cell HIV-1 Transmission, Resistance to Broadly Neutralizing Antibodies, and an Envelope Sorting Motif

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
S. Abigail Smith ◽  
Cynthia A. Derdeyn
Keyword(s):  
Neutralizing Antibodies ◽  
Differential Susceptibility ◽  
Neutralizing Antibody ◽  
Dependent Manner ◽  
Broadly Neutralizing Antibodies ◽  
Cell Infection ◽  
Viral Spread ◽  
Broadly Neutralizing Antibody ◽  
Hiv 1

ABSTRACT HIV-1 infection from cell-to-cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al. (H. Li, C. Zony, P. Chen, and B. K. Chen, J. Virol. 91:e02425-16, 2017, https://doi.org/10.1128/JVI.02425-16 ) showed that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell-to-cell infection in a context-dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

scholarly journals Identification of the natural product berberine as an antiviral drug

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jiping Shao ◽  
Debin Zeng ◽  
Shuhong Tian ◽  
Gezhi Liu ◽  
Jian Fu
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Antiviral Drug ◽  
Host Cells ◽  
Heptad Repeat ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Antiviral Activities ◽  
Hiv 1

Abstract Drugs targeting the fusion process of viral entry into host cells have been approved for clinical use in the treatment of AIDS. There remains a great need to improve the use of existing drugs for HIV therapy. Berberine is traditionally used to treat diarrhea, bacillary dysentery, and gastroenteritis in clinics, here our research shows that berberine is effective in inhibiting HIV-1 entry. Native polyacrylamide gel electrophoresis studies reveal that berberine can directly bind to both N36 and C34 to form a novel N36-berberine-C34 complex and effectively block the six-helix bundle formation between the N-terminal heptad repeat peptide N36 and the C-terminal heptad repeat peptide C34. Circular dichroism experiments show that binding of berberine produces conformational changes that damages the secondary structures of 6-HB. Computer-aided molecular docking studies suggest a hydrogen bond with T-639 and two polar bonds with Q-563 and T-639 are established, involving the oxygen atom and the C=O group of the indole ring. Berberine completely inhibits six HIV-1 clade B isolates and exhibits antiviral activities in a concentration-dependent manner with IC50 values varying from 5.5 to 10.25 µg/ml. This compound-peptide interaction may represent a mechanism of action of antiviral activities of berberine. As a summary, these studies successfully identify compound berberine as a potential candidate drug for HIV-1 treatment. As a summary, antiviral activity of berberine in combination with its use in clinical practice, this medicine can be used as a potential clinically anti-HIV drug.

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