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Antiviral determinants of rat Mx GTPases map to the carboxy-terminal half
L Johannes1, R Kambadur, H Lee-Hellmich
1Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, USA.
Abstract:
Rat Mx2 and rat Mx3 are two alpha/beta interferon-inducible cytoplasmic GTPases that differ in three residues in the amino-terminal third, which also contains the tripartite GTP-binding domain, and that differ in five residues in the carboxy-terminal quarter, which also contains a dimerization domain. While Mx2 is active against vesicular stomatitis virus (VSV), Mx3 lacks antiviral activity. We mapped the functional difference between Mx2 and Mx3 protein to two critical residues in the carboxy-terminal parts of the molecules. An exchange of either residue 588 or 630 of Mx2 with the corresponding residues of Mx3 abolished anti-VSV activity, and the introduction of the two Mx2 residues on an Mx3 background partially restored anti-VSV activity. These results are consistent with the facts that Mx2 and Mx3 have similar intrinsic GTPase activities and that the GTPase domain of Mx3 can fully substitute for the GTPase domain of Mx2. Nevertheless, the amino-terminal third containing the GTP-binding domain is necessary for antiviral activity, since an amino-terminally truncated Mx2 protein is devoid of anti-VSV activity. Furthermore, Fab fragments of a monoclonal antibody known to neutralize antiviral activity block GTPase activity by binding an epitope in the carboxy-terminal half of Mx2 or Mx3 protein. The results are consistent with a two-domain model in which both the conserved amino-terminal half and the less-well-conserved carboxy-terminal half of Mx proteins carry functionally important domains.
Insights
Rat Mx2 and Mx3 are interferon-inducible GTPases. Key differences in their carboxy-terminal regions determine Mx2
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Rat Mx2 and Mx3 are interferon-inducible cytoplasmic GTPases.
- These proteins share structural similarities, including a tripartite GTP-binding domain and a dimerization domain.
- Mx2 exhibits antiviral activity against vesicular stomatitis virus (VSV), while Mx3 lacks this activity.
Purpose of the Study:
- To map the functional difference in antiviral activity between rat Mx2 and Mx3 proteins.
- To investigate the role of specific residues and domains in the antiviral function of Mx proteins.
Main Methods:
- Site-directed mutagenesis was employed to exchange specific amino acid residues between Mx2 and Mx3.
- Antiviral activity against VSV was assessed for wild-type and mutant Mx proteins.
- GTPase activity was measured, and antibody-binding assays were performed using Fab fragments.
Main Results:
- The functional difference between Mx2 and Mx3 was mapped to two critical residues (588 and 630) in the carboxy-terminal region.
- Exchanging either residue 588 or 630 in Mx2 with the corresponding Mx3 residue abolished anti-VSV activity.
- Introducing the two Mx2 residues into Mx3 partially restored antiviral activity, indicating their crucial role.
- The GTP-binding domain is essential for antiviral activity, as an amino-terminally truncated Mx2 protein lost its function.
- Antibody fragments that neutralize antiviral activity bind to an epitope in the carboxy-terminal half, inhibiting GTPase activity.
Conclusions:
- The carboxy-terminal region, specifically residues 588 and 630, is critical for the differential antiviral activity of rat Mx2 and Mx3.
- Both the amino-terminal GTP-binding domain and the carboxy-terminal region are functionally important for Mx protein antiviral activity.
- A two-domain model is proposed, where both conserved and less-conserved regions of Mx proteins contribute to their function.