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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

Theoretical structures of GRFT-tandemers. Griffithsin tandemers 2mGRFT, 2mGRFT3, 3mGRFT, and 4mGRFT are shown as models of monomeric units attached by flexible linker regions. The N and C termini (red and black, respectively) in a single mGRFT domain are approximately 10 Å apart, causing the individual domains in the tandemers to branch out. Each Gly-Thr-Gly linker (blue) is also approximately 10 Å long in its extended conformation.
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Fig2: Theoretical structures of GRFT-tandemers. Griffithsin tandemers 2mGRFT, 2mGRFT3, 3mGRFT, and 4mGRFT are shown as models of monomeric units attached by flexible linker regions. The N and C termini (red and black, respectively) in a single mGRFT domain are approximately 10 Å apart, causing the individual domains in the tandemers to branch out. Each Gly-Thr-Gly linker (blue) is also approximately 10 Å long in its extended conformation.

Mentions: The GRFT monomer 1GS-S ([30], PDB ID 3LL2, Figure 1B) was chosen as the repeating unit in the design of mGRFT tandemers, because the L2S mutation at its N terminus rendered this monomer more susceptible to proteolytic cleavage of its N-terminal affinity tag. All further references to mGRFT in this work are to 1GS-S. Schematic models of mGRFT tandemers constructed based on the structure of mGRFT [30] are depicted in Figure 2. The linker between each mGRFT domain was chosen to be flexible and unstructured and consisted of single and triple repeats of Gly-Thr-Gly. However, the flexibility of the linker rendered all attempts at crystallizing the tandemers unsuccessful. Purification of tandemers was performed largely as described previously [30]. The relevant information for GRFT, mGRFT, and the tandemers is listed in Table 1. The relative thermal stability of the mGRFT tandemers was evaluated by differential scanning calorimetry (Additional file 1: Table S1). It was found that the mGRFT tandemers have similar melting temperatures to mGRFT, indicating that the lectin domains were properly folded.Figure 2


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)

Theoretical structures of GRFT-tandemers. Griffithsin tandemers 2mGRFT, 2mGRFT3, 3mGRFT, and 4mGRFT are shown as models of monomeric units attached by flexible linker regions. The N and C termini (red and black, respectively) in a single mGRFT domain are approximately 10 Å apart, causing the individual domains in the tandemers to branch out. Each Gly-Thr-Gly linker (blue) is also approximately 10 Å long in its extended conformation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Theoretical structures of GRFT-tandemers. Griffithsin tandemers 2mGRFT, 2mGRFT3, 3mGRFT, and 4mGRFT are shown as models of monomeric units attached by flexible linker regions. The N and C termini (red and black, respectively) in a single mGRFT domain are approximately 10 Å apart, causing the individual domains in the tandemers to branch out. Each Gly-Thr-Gly linker (blue) is also approximately 10 Å long in its extended conformation.
Mentions: The GRFT monomer 1GS-S ([30], PDB ID 3LL2, Figure 1B) was chosen as the repeating unit in the design of mGRFT tandemers, because the L2S mutation at its N terminus rendered this monomer more susceptible to proteolytic cleavage of its N-terminal affinity tag. All further references to mGRFT in this work are to 1GS-S. Schematic models of mGRFT tandemers constructed based on the structure of mGRFT [30] are depicted in Figure 2. The linker between each mGRFT domain was chosen to be flexible and unstructured and consisted of single and triple repeats of Gly-Thr-Gly. However, the flexibility of the linker rendered all attempts at crystallizing the tandemers unsuccessful. Purification of tandemers was performed largely as described previously [30]. The relevant information for GRFT, mGRFT, and the tandemers is listed in Table 1. The relative thermal stability of the mGRFT tandemers was evaluated by differential scanning calorimetry (Additional file 1: Table S1). It was found that the mGRFT tandemers have similar melting temperatures to mGRFT, indicating that the lectin domains were properly folded.Figure 2

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