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Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus.

Moulaei T, Alexandre KB, Shenoy SR, Meyerson JR, Krumpe LR, Constantine B, Wilson J, Buckheit RW, McMahon JB, Subramaniam S, Wlodawer A, O'Keefe BR - Retrovirology (2015)

Bottom Line: Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM.Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity.We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions.

View Article: PubMed Central - PubMed

Affiliation: Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702-1201, USA. tinoush@umd.edu.

ABSTRACT

Background: The lectin griffithsin (GRFT) is a potent antiviral agent capable of prevention and treatment of infections caused by a number of enveloped viruses and is currently under development as an anti-HIV microbicide. In addition to its broad antiviral activity, GRFT is stable at high temperature and at a broad pH range, displays little toxicity and immunogenicity, and is amenable to large-scale manufacturing. Native GRFT is a domain-swapped homodimer that binds to viral envelope glycoproteins and has displayed mid-picomolar activity in cell-based anti-HIV assays. Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM. Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity.

Results: Here we present data on engineered tandem repeats of mGRFT (mGRFT tandemers) with antiviral activity at concentrations as low as one picomolar in whole-cell anti-HIV assays. mGRFT tandemers were analyzed thermodynamically, both individually and in complex with HIV-1 gp120. We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions. This establishes that, although the intra-virion crosslinking of HIV envelope glycoproteins is likely integral to their activity, the antiviral activity of these lectins is not due to virus aggregation caused by inter-virion crosslinking.

Conclusions: The engineered tandemer constructs of mGRFT may provide novel and powerful agents for prevention of infection by HIV and other enveloped viruses.

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Related in: MedlinePlus

Electron micrographs of the HIV-1 virions interacting with different constructs of GRFT-based lectins. (A) Projection of untreated HIV-1 virions. Virions are observed as ~ 100 nm circles in the imaging field which includes 10-nanometer-sized protein-A gold fiducials which appear as dark spots. (B, C) Slices through tomograms of untreated HIV-1 virions with red arrows indicating individual Env proteins. (D) Projection of HIV-1 virions that were incubated with mGRFT prior to vitrification. (E, F) Slices through tomograms of HIV-1 virions treated with mGRFT. Representative Env glycoprotein spikes are indicated by red arrows (E). A patch of glycoprotein spikes is indicated by a red arc (F). (G) Projection of HIV-1 virions that were incubated with native GRFT prior to vitrification. (H, I) Slices through tomograms of virions at the periphery of the aggregates in virions treated with native GRFT. (J–M) Slices through tomograms collected from vitreous preparations of HIV-1 virions with GRFT tandemer constructs, 2mGRFT (J), 2mGRFT3 (K), 3mGRFT (I), and 4mGRFT (M). Scale bars are 500 nm in panels (A, D, and G) and 50 nm in the remaining panels.
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Fig5: Electron micrographs of the HIV-1 virions interacting with different constructs of GRFT-based lectins. (A) Projection of untreated HIV-1 virions. Virions are observed as ~ 100 nm circles in the imaging field which includes 10-nanometer-sized protein-A gold fiducials which appear as dark spots. (B, C) Slices through tomograms of untreated HIV-1 virions with red arrows indicating individual Env proteins. (D) Projection of HIV-1 virions that were incubated with mGRFT prior to vitrification. (E, F) Slices through tomograms of HIV-1 virions treated with mGRFT. Representative Env glycoprotein spikes are indicated by red arrows (E). A patch of glycoprotein spikes is indicated by a red arc (F). (G) Projection of HIV-1 virions that were incubated with native GRFT prior to vitrification. (H, I) Slices through tomograms of virions at the periphery of the aggregates in virions treated with native GRFT. (J–M) Slices through tomograms collected from vitreous preparations of HIV-1 virions with GRFT tandemer constructs, 2mGRFT (J), 2mGRFT3 (K), 3mGRFT (I), and 4mGRFT (M). Scale bars are 500 nm in panels (A, D, and G) and 50 nm in the remaining panels.

Mentions: The interactions of GRFT, mGRFT, and the tandemers with purified HIV-1BaL virions were evaluated using cryo-electron microscopy. The control experiments with HIV-1Bal in the absence of any lectins showed a uniform distribution of virions in the imaging field, with virions having approximately 100 nm diameter and spherical shape (Figure 5A). Ten-nanometer gold fiducials appear as electron dense spots in the micrographs. When virions were imaged at high magnification with cryo-electron tomography, individual envelope glycoprotein spikes were visible (Figures 5B, C, red arrows). The spikes were of the expected height of ~ 120 Å as measured from the membrane, with a structural profile consistent with previous electron tomographic studies [33-38].Figure 5


Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus.

Moulaei T, Alexandre KB, Shenoy SR, Meyerson JR, Krumpe LR, Constantine B, Wilson J, Buckheit RW, McMahon JB, Subramaniam S, Wlodawer A, O'Keefe BR - Retrovirology (2015)

Electron micrographs of the HIV-1 virions interacting with different constructs of GRFT-based lectins. (A) Projection of untreated HIV-1 virions. Virions are observed as ~ 100 nm circles in the imaging field which includes 10-nanometer-sized protein-A gold fiducials which appear as dark spots. (B, C) Slices through tomograms of untreated HIV-1 virions with red arrows indicating individual Env proteins. (D) Projection of HIV-1 virions that were incubated with mGRFT prior to vitrification. (E, F) Slices through tomograms of HIV-1 virions treated with mGRFT. Representative Env glycoprotein spikes are indicated by red arrows (E). A patch of glycoprotein spikes is indicated by a red arc (F). (G) Projection of HIV-1 virions that were incubated with native GRFT prior to vitrification. (H, I) Slices through tomograms of virions at the periphery of the aggregates in virions treated with native GRFT. (J–M) Slices through tomograms collected from vitreous preparations of HIV-1 virions with GRFT tandemer constructs, 2mGRFT (J), 2mGRFT3 (K), 3mGRFT (I), and 4mGRFT (M). Scale bars are 500 nm in panels (A, D, and G) and 50 nm in the remaining panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4419512&req=5

Fig5: Electron micrographs of the HIV-1 virions interacting with different constructs of GRFT-based lectins. (A) Projection of untreated HIV-1 virions. Virions are observed as ~ 100 nm circles in the imaging field which includes 10-nanometer-sized protein-A gold fiducials which appear as dark spots. (B, C) Slices through tomograms of untreated HIV-1 virions with red arrows indicating individual Env proteins. (D) Projection of HIV-1 virions that were incubated with mGRFT prior to vitrification. (E, F) Slices through tomograms of HIV-1 virions treated with mGRFT. Representative Env glycoprotein spikes are indicated by red arrows (E). A patch of glycoprotein spikes is indicated by a red arc (F). (G) Projection of HIV-1 virions that were incubated with native GRFT prior to vitrification. (H, I) Slices through tomograms of virions at the periphery of the aggregates in virions treated with native GRFT. (J–M) Slices through tomograms collected from vitreous preparations of HIV-1 virions with GRFT tandemer constructs, 2mGRFT (J), 2mGRFT3 (K), 3mGRFT (I), and 4mGRFT (M). Scale bars are 500 nm in panels (A, D, and G) and 50 nm in the remaining panels.
Mentions: The interactions of GRFT, mGRFT, and the tandemers with purified HIV-1BaL virions were evaluated using cryo-electron microscopy. The control experiments with HIV-1Bal in the absence of any lectins showed a uniform distribution of virions in the imaging field, with virions having approximately 100 nm diameter and spherical shape (Figure 5A). Ten-nanometer gold fiducials appear as electron dense spots in the micrographs. When virions were imaged at high magnification with cryo-electron tomography, individual envelope glycoprotein spikes were visible (Figures 5B, C, red arrows). The spikes were of the expected height of ~ 120 Å as measured from the membrane, with a structural profile consistent with previous electron tomographic studies [33-38].Figure 5

Bottom Line: Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM.Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity.We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions.

View Article: PubMed Central - PubMed

Affiliation: Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, 21702-1201, USA. tinoush@umd.edu.

ABSTRACT

Background: The lectin griffithsin (GRFT) is a potent antiviral agent capable of prevention and treatment of infections caused by a number of enveloped viruses and is currently under development as an anti-HIV microbicide. In addition to its broad antiviral activity, GRFT is stable at high temperature and at a broad pH range, displays little toxicity and immunogenicity, and is amenable to large-scale manufacturing. Native GRFT is a domain-swapped homodimer that binds to viral envelope glycoproteins and has displayed mid-picomolar activity in cell-based anti-HIV assays. Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM. Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity.

Results: Here we present data on engineered tandem repeats of mGRFT (mGRFT tandemers) with antiviral activity at concentrations as low as one picomolar in whole-cell anti-HIV assays. mGRFT tandemers were analyzed thermodynamically, both individually and in complex with HIV-1 gp120. We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions. This establishes that, although the intra-virion crosslinking of HIV envelope glycoproteins is likely integral to their activity, the antiviral activity of these lectins is not due to virus aggregation caused by inter-virion crosslinking.

Conclusions: The engineered tandemer constructs of mGRFT may provide novel and powerful agents for prevention of infection by HIV and other enveloped viruses.

Show MeSH
Related in: MedlinePlus