Vesicular stomatitis virus transcription inhibited by purified MxA protein.
MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammalian cells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTP gamma S, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.
Schwemmle
M
M
Abteilung Virologie, Universität Freiburg, Germany.
Weining
K C
KC
Richter
M F
MF
Schumacher
B
B
Staeheli
P
P
D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D064547:Myxovirus Resistance Proteins; D009711:Nucleotides / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000502:physiology*; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*
D000998:Antiviral Agents; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D009711:Nucleotides; D011506:Proteins; D006639:Histidine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins