guanosine-triphosphate and Orthomyxoviridae-Infections

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Topics: Animals; Cell Compartmentation; Cell Line; Cell Polarity; Cell Survival; DNA-Directed RNA Polymerases; Dogs; Endocytosis; Endosomes; Guanosine Triphosphate; Immunoprecipitation; Influenza A virus; Microtubules; Models, Biological; Orthomyxoviridae Infections; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Protein Transport; rab GTP-Binding Proteins; Ribonucleoproteins; Signal Transduction

The murine Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection even though the resistance mechanism is yet unclear. The Mx1 protein contains a tripartite GTP-binding domain consisting of GXXXXGKS, DXXG, and T/NKXD motifs. In the GTPase gene superfamily such as p21ras protein, signal-transducing G protein, and translation elongation factor, the GTPase activity plays a key role in each protein function. Here we show that GTPase activity is indeed associated with the intact Mx1 protein purified from Escherichia coli expressing Mx1 cDNA. Amino acid substitution within the GTP-binding motif led to significant reduction in the GTPase activity. Yeast vacuolar protein sorting (VPS1) protein and the rat microtubule-associated mechanochemical enzyme dynamin were found to be homologous to Mx1 not only in the tripartite GTP-binding motif, but also in the amino-terminal region of approximately 300 amino acids in length. The function of Mx1 is discussed in comparison with these proteins.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Base Sequence; Drug Resistance; Escherichia coli; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Kinetics; Mice; Molecular Sequence Data; Multigene Family; Mutagenesis, Site-Directed; Myxovirus Resistance Proteins; Oligodeoxyribonucleotides; Orthomyxoviridae Infections; Protein Biosynthesis; Proteins; Recombinant Proteins