A monomeric GTPase-negative MxA mutant with antiviral activity - PubMed (original) (raw)

A monomeric GTPase-negative MxA mutant with antiviral activity

C Janzen et al. J Virol. 2000 Sep.

Abstract

MxA is a large, interferon-induced GTPase with antiviral activity against RNA viruses. It forms large oligomers, but whether oligomerization and GTPase activity are important for antiviral function is not known. The mutant protein MxA(L612K) carries a lysine-for-leucine substitution at position 612 and fails to form oligomers. Here we show that monomeric MxA(L612K) lacks detectable GTPase activity but is capable of inhibiting Thogoto virus in transiently transfected Vero cells or in a Thogoto virus minireplicon system. Likewise, MxA(L612K) inhibited vesicular stomatitis virus multiplication. These findings indicate that MxA monomers are antivirally active and suggest that GTP hydrolysis may not be required for antiviral activity. MxA(L612K) is rapidly degraded in cells, whereas wild-type MxA is stable. We propose that high-molecular-weight MxA oligomers represent a stable intracellular pool from which active MxA monomers are recruited.

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Figures

FIG. 1

FIG. 1

MxA(L612K) lacks detectable GTPase activity. GTP hydrolysis of mutant MxA(L612K) protein was compared with that of wild-type MxA [MxA(wt)] or inactive mutant MxA(T103A) in a standard GTPase assay. Lane 1 is a control without protein in the reaction mixture.

FIG. 2

FIG. 2

MxA(L612K) is antivirally active. (A) Vero cells transfected with cDNAs encoding either wild-type MxA [MxA(wt)] or mutant protein MxA(L612K) or MxA(T103A) were infected with 50 PFU of THOV per cell. Cells were fixed 9 h later, and MxA proteins or viral proteins were detected by double immunofluorescence using a monoclonal antibody directed against MxA and a polyclonal antiserum directed against THOV antigens. (B) Quantitative analysis of infection experiments with THOV and VSV. The percentage of infected cells expressing either wild-type MxA [MxA(wt)], MxA(L612K), or MxA(T103A) is shown. Cells were infected with 10 PFU of VSV serotype Indiana per cell (15).

FIG. 3

FIG. 3

MxA(L612K) inhibits reporter gene expression in a THOV minireplicon system. COS-1 cells were transfected with T7 promoter constructs coding for the components of a THOV minireplicon system as previously described (29). In this system, synthesis of CAT protein reflects the activity of the reconstituted vRNPs (30). Wild-type or mutant MxA was expressed under the control of the T7 promoter using increasing amounts of expression plasmid pBS-T7/MxA, pBS-T7/MxA(L612K), or pBS-T7/MxA(T103A). (A) CAT protein concentration as determined by a colorimetric immunoassay (Boehringer Mannheim). The amounts of CAT and luciferase activity were determined in the cell lysates. Luciferase activity was used to normalize CAT expression, and the ratio of CAT protein concentration to luciferase activity (CAT/Luc ratio) was calculated as described previously (29). The CAT/luciferase ratio of experiments without MxA were set at 1. (B) Expression of MxA proteins. Aliquots of the cell lysates (15 μg of protein per lane) were analyzed by Western blotting using a polyclonal antiserum directed against MxA.

FIG. 4

FIG. 4

MxA(L612K) is rapidly degraded. COS-1 cells were transfected with expression plasmids coding for either MxA or MxA(L612K). Cells were then labeled for 2 h with 35 μCi of [35S]methionine per ml and chased for 2.5, 5, 7.5, and 10 h. (A) Wild-type and mutant MxA proteins were immunoprecipitated from cell lysates using a monoclonal antibody directed against MxA and analyzed by polyacrylamide gel electrophoresis and autoradiography. (B) PhosphoImager analysis of immunoprecipitated proteins. MxA(wt), wild-type MxA.

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