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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

Location of GRFT binding-associated oligosaccharides (coupled to Asn234, Asn295, and Asn448) in the context of HIV trimeric spike. Disaccharide modification of Asn234 (blue), Asn295 (red) and Asn448 (yellow) are displayed on a gp120 trimer. X-ray crystal structure of the partially glycosylated HIV-1HXBc2 gp120 (PDB 1GC1 [45]) was superimposed on unliganded gp120 electron tomography structure (PDB 3DNN [34]).
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Fig6: Location of GRFT binding-associated oligosaccharides (coupled to Asn234, Asn295, and Asn448) in the context of HIV trimeric spike. Disaccharide modification of Asn234 (blue), Asn295 (red) and Asn448 (yellow) are displayed on a gp120 trimer. X-ray crystal structure of the partially glycosylated HIV-1HXBc2 gp120 (PDB 1GC1 [45]) was superimposed on unliganded gp120 electron tomography structure (PDB 3DNN [34]).

Mentions: These observations have implications for understanding the mechanism whereby the mGRFT tandemers neutralize HIV. The antiviral activity of GRFT, mGRFT, and the mGRFT tandemers stems from their selective binding to high mannose oligosaccharides on Env spikes. Natural mutations that removed glycosylation at positions Asn234 and Asn295 have been reported to impart resistance to GRFT [43]. In addition, Huang et al. showed that deglycosylation of gp120 at Asn295 or Asn448 also resulted in resistance to GRFT [44]. The location of these oligosaccharide attachment sites on gp120 are shown in Figure 6. To investigate the role of specific oligosaccharides in the sensitivity to GRFT tandemers, we utilized genetically-engineered pseudotype viruses including either HIV-1 clade B CAAN5342 gp120 (Figure 3A) or clade C CAP206.8 gp120 (Figure 3B) sequences. We tested both strains in either the presence or absence of oligosaccharide attachment sites at positions 234 N and 295 N of the gp120 protein. Figure 3 displays the results of our assessment of the activity of either wild-type GRFT, mGRFT, or mGRFT tandemers against these viruses. The 3mGRFT and 4mGRFT tandemers retain significantly more activity against wild-type GRFT-resistant viruses than the 2mGRFT, 2mGRFT3, or wild-type GRFT. We speculate that this increased activity against resistant viruses is due to the ability of these larger mGRFT tandemers to sample a broader range of oligosaccharides on gp120 than the smaller mGRFT tandemers or the conformationally-restricted wild-type GRFT.Figure 6


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)

Location of GRFT binding-associated oligosaccharides (coupled to Asn234, Asn295, and Asn448) in the context of HIV trimeric spike. Disaccharide modification of Asn234 (blue), Asn295 (red) and Asn448 (yellow) are displayed on a gp120 trimer. X-ray crystal structure of the partially glycosylated HIV-1HXBc2 gp120 (PDB 1GC1 [45]) was superimposed on unliganded gp120 electron tomography structure (PDB 3DNN [34]).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Location of GRFT binding-associated oligosaccharides (coupled to Asn234, Asn295, and Asn448) in the context of HIV trimeric spike. Disaccharide modification of Asn234 (blue), Asn295 (red) and Asn448 (yellow) are displayed on a gp120 trimer. X-ray crystal structure of the partially glycosylated HIV-1HXBc2 gp120 (PDB 1GC1 [45]) was superimposed on unliganded gp120 electron tomography structure (PDB 3DNN [34]).
Mentions: These observations have implications for understanding the mechanism whereby the mGRFT tandemers neutralize HIV. The antiviral activity of GRFT, mGRFT, and the mGRFT tandemers stems from their selective binding to high mannose oligosaccharides on Env spikes. Natural mutations that removed glycosylation at positions Asn234 and Asn295 have been reported to impart resistance to GRFT [43]. In addition, Huang et al. showed that deglycosylation of gp120 at Asn295 or Asn448 also resulted in resistance to GRFT [44]. The location of these oligosaccharide attachment sites on gp120 are shown in Figure 6. To investigate the role of specific oligosaccharides in the sensitivity to GRFT tandemers, we utilized genetically-engineered pseudotype viruses including either HIV-1 clade B CAAN5342 gp120 (Figure 3A) or clade C CAP206.8 gp120 (Figure 3B) sequences. We tested both strains in either the presence or absence of oligosaccharide attachment sites at positions 234 N and 295 N of the gp120 protein. Figure 3 displays the results of our assessment of the activity of either wild-type GRFT, mGRFT, or mGRFT tandemers against these viruses. The 3mGRFT and 4mGRFT tandemers retain significantly more activity against wild-type GRFT-resistant viruses than the 2mGRFT, 2mGRFT3, or wild-type GRFT. We speculate that this increased activity against resistant viruses is due to the ability of these larger mGRFT tandemers to sample a broader range of oligosaccharides on gp120 than the smaller mGRFT tandemers or the conformationally-restricted wild-type GRFT.Figure 6

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